JP5919192B2 - Preparation of β-amino acids - Google Patents

Description

  The present invention relates to a process for biocatalytically producing β-amino acid precursors from optionally substituted or unsubstituted dihydrouracil using hydantoinase and / or dihydropyrimidinase, β-amino acids from said precursors The present invention relates to a hydantoinase and its use in the process for producing a β-amino acid precursor or β-amino acid with a biocatalyst, and a method for obtaining the hydantoinase.

  Enantiomerically pure β-amino acids are an important component for new therapeutic agents with a wide range of biological activities. Several biocatalytic pathways have been developed to prepare this, but there is still no single method that is widely applicable in general. Similarly, a slightly catalyzed route to β-amino acids has been developed, but most require a stoichiometric amount of chiral auxiliary.

  Dihydropyrimidinase and hydantoinase are potential candidates for biocatalytic synthesis of amino acids or their precursors. Gaebler and Keltch first reported hydantoinase cleavage activity in the 1920s (Gaebler, O. H .; Keltch, A. K. On the metabolism of hydantoins and hydantoic acid, 1926; Vol. 70). Initially suggested by Eadie et al. In the 1950s, microbial hydantoinase was the same as animal dihydropyrimidinase (Eadie, G .; Bernheim, F .; Bernheim, M. Journal of Biological Chemistry 181: 449-458, 1949). Dihydropyrimidinase enzymes isolated from calf liver and plants catalyze the hydrolysis of dihydrouracil and dihydrothymine to give N-carbamoyl-β-alanine and N-carbamoyl-2-methyl-β-alanine, respectively. It was. The enzyme also cleaves (R) -5-monosubstituted hydantoins to (R) -N-carbamoyl amino acids. Recent literature generally suggests that D-hydantoinase of microbial origin can be regarded as the equivalent of animal dihydropyrimidinase, and Nonaka and its collaborators This suggests an evolutionary relationship (Hamajima, N .; Matsuda, K .; Sakata, S .; Tamaki, N .; Sasaki, M .; Nonaka, M. Gene 180: 157-163, 1996). Syldatk et al. Conclude that dihydropyrimidinase and hydantoinase are not necessarily the same enzyme (Syldatk, C .; May, O .; Altenbuchner, J .; Mattes, R .; Siemann, M. Applied Microbiology and Biotechnology 51 : 293-309, 1999). Differences in the enantioselectivity of hydantoinases are often used to classify them as D-, L-, or non-specific according to specificity (Ogawa, J .; Shimizu, S. Journal of Molecular Catalysis B: Enzymatic 2: 163-176, 1997).

  The problem due to the naming system used for hydantoinase and dihydropyrimidinase enzymes is exacerbated by the fact that the terms were used indistinguishable from each other, especially in early academic journals. Amidohydrolases, also called cyclic amidases [EC3.5.2], are a group of 15 or more enzymes, all of which act on the cyclic amide ring and are in many highly conserved regions, And has an invariant amino acid region (Kim, GJ; Cheon, YH; Kim, HS Biotechnology and Bioengineering 1998, 61, 1-13). This group of enzymes includes carboxymethylhydantoinase [EC3.5.2.4], allantoinase [EC3.5.2.5], 1-methylhydantoinase [EC3.5.2.14], and carboxyethylhydantoinase. All of these are scientifically only one hydantoinase because the substrate is a naturally occurring hydantoin derivative.

  Other enzymes that fall into the broader class of cyclic amidases include dihydroorotase [EC3.5.2.3] and dihydropyrimidinase [EC3.5.2.2], the latter being (R) -5-monosubstituted hydantoins Because of its ability to hydrolyze derivatives, it is usually called D-hydantoinase. This superfamily of proteins probably evolved on prehistoric Earth, assuming that the N-carbamoyl amino acid was the first synthon of the prebiotic peptide.

  For enantioselective hydrolysis of racemic mixtures of 5-substituted hydantoins (R) -1 and (S) -1 to the corresponding N-carbamoyl derivatives (R) -2 and (S) -2 The use of hydantoinase is well established (see Scheme 1 below) and is described in the literature (Morin, Enzyme Microb. Technol. 15: 208-214, 1993; Fan and Lee, Biochemical Engineering J. 8: 157-164, 2001; Arcuri et al., J. Molecular Catalysis B 21: 107-111, 2003; Arcuri et al., Amino Acids 19: 477-482, 2000). It has reached a stage where an industrial process operates on a scale to produce specific D- (R) -amino acids (R) -3 using this technology. An important aspect of these processes is the in situ racemization of enantiomer (S) -1 as well as the hydrolysis of (R) -2 catalyzed by carbamoylase, which is dynamic kinetic optical resolution (DKR) ) Bring reaction.

(Scheme 1)

  If the enzyme rotates one enantiomer faster than the other, kinetic resolution occurs. However, the maximum yield of this type of reaction is only 50% and the product needs to be separated from the starting material. In dynamic kinetic resolution, the enantiomers racemize and the (R)-and (S) -enantiomers are in chemical equilibrium and readily interconvert. When a rapidly reacting enantiomer is converted to the corresponding product, racemization supplements the enantiomer, allowing yields up to 100%.

  In contrast to enantioselective hydrolysis of racemic 5-substituted hydantoins, as a route to β-amino acids (R or S) -6, 6-substituted dihydrouracil (+/-)-4 (see Scheme 2) The possibility of performing enantioselective hydrolysis to the corresponding N-carbamoyl derivative (R or S) -5 received little attention. Syldatk et al. In 1998 (May, O .; Siemann, M .; Pietzsch, M .; Kiess, M .; Mattes, R .; Syldatk, CJ Biotechnol. 61: 1-13, 1998), dihydrouracil ( Reported the use of Hydantoinase from Arthrobacter aurescens for the hydrolysis of (+/-)-4, where R represents H), and in 2003, in a poster, 5-phenylhydantoin ((R) -1 And (S) -1, where R represents Ph in Scheme 1, respectively, although the enantioselectivity was weak and the reaction rate was observed to be low, this hydantoinase was converted to 6-phenyldihydrouracil (6-PDHU, ( +/-)-4, where R represents phenyl).

(Scheme 2)

  Japanese patent application JP06261787 reported up to 51% enantiomeric excess for hydrolysis of 6-PDHU using Pseudomonas putida IFO 12996; better selectivity (up to 93% for substrates with 6-alkyl substituents) (Enantiomer excess). Clearly there is a need for improved methods for the production of β-amino acid precursors or β-amino acids by biocatalysis.

Japanese patent application JP06261787

Gaebler, O. H .; Keltch, A. K. On the metabolism of hydantoins and hydantoic acid, 1926; Vol. 70 Eadie, G .; Bernheim, F .; Bernheim, M. Journal of Biological Chemistry 181: 449-458, 1949 Hamajima, N .; Matsuda, K .; Sakata, S .; Tamaki, N .; Sasaki, M .; Nonaka, M. Gene 180: 157-163, 1996 Syldatk, C .; May, O .; Altenbuchner, J .; Mattes, R .; Siemann, M. Applied Microbiology and Biotechnology 51: 293-309, 1999 Ogawa, J .; Shimizu, S. Journal of Molecular Catalysis B: Enzymatic 2: 163-176, 1997 Kim, G. J .; Cheon, Y. H .; Kim, H. S. Biotechnology and Bioengineering 1998, 61, 1-13 Morin, Enzyme Microb. Technol. 15: 208-214, 1993; Fan and Lee, Biochemical Engineering J. 8: 157-164, 2001; Arcuri et al., J. Molecular Catalysis B 21: 107-111, 2003; Arcuri et al., Amino Acids 19: 477-482, 2000 May, O .; Siemann, M .; Pietzsch, M .; Kiess, M .; Mattes, R .; Syldatk, C. J. Biotechnol. 61: 1-13, 1998

(Brief description of the invention)
In a first aspect, the object of the present invention has been solved by a process for producing a β-amino acid precursor stereospecifically, more particularly enantioselectively with a biocatalyst, said process comprising at least One general formula (I)

[Wherein R ¹ and R ² are independently of each other hydrogen;
A linear or branched lower alkyl group which may be substituted;
A linear or branched lower alkenyl group which may be substituted;
A cyclic alkyl group which may be substituted;
An optionally substituted monocyclic or polycyclic aryl group;
An optionally substituted monocyclic or polycyclic heteroaryl group;
A linear or branched alkoxy group which may be substituted;
An amino group;
A linear or branched alkylamino group which may be substituted;
A linear or branched alkylthio group which may be substituted;
A linear or branched acyl group which may be substituted;
Selected from carboxyl group or aldehyde group]
A substrate represented by
Stereoisomerically pure forms, such as (R)-or (S) -isomers, or mixtures of stereoisomers, such as racemic mixtures,
Or a salt of said compound, for example an acid addition salt;
The substrate in the form of
Selected from at least one enzyme that catalyzes the hydrolysis of the hydantoin and / or dihydropyrimidine ring, in particular hydantoinase and dihydropyrimidinase, more particularly a hydantoinase, in particular a compound to be converted In the presence of an enzyme that has a preference for a particular isomer of; and optionally in the presence of at least one enzyme capable of interconverting the stereoisomer of said compound of general formula (I) React
As a result, β-amino acid precursor of general formula (II)

[Wherein R ¹ and R ² are the same as above]
Generating;
A process comprising:
Said process wherein at least one enzyme catalyzing the hydrolysis of the hydantoin and / or dihydropyrimidine ring, in particular hydantoinase and / or dihydropyrimidinase is obtained from azuki bean (Vigna angularis), and / or Containing at least one partial sequence having about 60 to 100% identity to at least one of the following partial sequences;
ITGPEGQRLAGP (SEQ ID NO: 7)
IELGITGPEGQRLAGPTVL (SEQ ID NO: 1)
IELGITGPEGQRLAGPVL (SEQ ID NO: 2)
IELITGPEGQRLAGPTVL (SEQ ID NO: 3)
IELITGPEGQRLAGPVL (SEQ ID NO: 4)
EEIARARKSGQRVIGEPVAS (SEQ ID NO: 5),
For example, it is characterized in that it contains at least one partial sequence having 60% to 100% identity to at least one of the partial sequences of SEQ ID NOs: 5 and 7.

According to certain embodiments, the process further comprises an additional step by converting said beta amino acid precursor of formula (2) to the corresponding beta amino acid of formula (III),

[Wherein R ¹ and R ² have the same meaning as described above].

  According to other embodiments, the conversion of the β-amino acid precursor occurs at acidic pH, preferably in the presence of nitrite or in the presence of carbamoylase.

  According to another embodiment, the at least one hydantoinase and / or dihydropyrimidinase (especially an enzyme of EC 3.5.2.2) is Agrobacterium, Arthrobacter, Pseudomonas and cowpea. It is an enzyme obtained from organisms of the genus (Vigna), in particular from azuki (Vigna angularis).

According to another embodiment, in general formulas (I) to (III), R ² is H and R ¹ is different from H.

According to another embodiment, in general formulas (I) to (III), R ² is H and R ¹ is an optionally substituted aryl group.

In particular, the compounds of formulas (II) and (III), as well as the compounds of formula (I), also have R ² being H, but R ¹ is different from H, so as (S) isomer Exist in a pure isomeric form or in a stereoisomeric excess (specifically having an ee value greater than 93%, preferably in the range of 95-99% or greater than 99%). preferable.

  According to other embodiments, the reaction is carried out in a reaction mixture buffered with Tris buffer or borate buffer, with a Tris buffer reaction mixture being preferred.

  According to other embodiments, the reaction is carried out at a pH of about 7.0 to about 11.0, but preferably has a pH of about 7.5 to about 10.0, with a pH of about 7.5 to about 8.0 being particularly preferred.

  According to another embodiment, the reaction is carried out in the presence of about 1% to about 20% dimethyl sulfoxide, but is preferably carried out in the presence of about 10% dimethyl sulfoxide.

  According to another embodiment, the reaction is carried out at a temperature in the range from about 30 ° C. to about 60 ° C., preferably at a temperature in the range from about 30 ° C. to about 50 ° C., in particular from about 40 ° C. to about 50 ° C. .

  According to another embodiment, the reaction is carried out from about 1 hour to about 25 hours, but is preferably carried out from about 3 hours to about 10 hours, especially from about 4 hours to about 5 hours.

  According to another embodiment, the at least one substrate is dihydrouracil monosubstituted at the 5 or 6 position, in particular 6-phenyldihydrouracil, 6- (4-fluorophenyl) -dihydrouracil, 6- (4 -Chlorophenyl) -dihydrouracil, 5-methyldihydrouracil, and 6-methyldihydrouracil.

  Another embodiment of the present invention is a pharmaceutically active agent which is a peptide that is stable against hydrolysis, in particular antibiotics, anticancer agents, antithrombotic agents, antifungal agents, insecticides, anthelmintics, non-peptidic agents It relates to the use of β-amino acid precursors or β-amino acids obtained by the process of the invention for the production of integrin antagonists, alkaloids and / or cytotoxic drugs.

Another embodiment of the present invention is at least one of the following partial sequences:
ITGPEGQRLAGP (SEQ ID NO: 7)
IELGITGPEGQRLAGPTVL (SEQ ID NO: 1)
IELGITGPEGQRLAGPVL (SEQ ID NO: 2)
IELITGPEGQRLAGPTVL (SEQ ID NO: 3)
IELITGPEGQRLAGPVL (SEQ ID NO: 4)
EEIARARKSGQRVIGEPVAS (SEQ ID NO: 5),
For example, at least one of the partial sequences of SEQ ID NOs: 5 and 7,
A hydantoinase and / or dihydropyrimidinase containing at least one subsequence having 60 to 100 percent identity to the use of the process of the present invention.

Another embodiment of the present invention is at least one of the following partial sequences:
ITGPEGQRLAGP (SEQ ID NO: 7)
IELGITGPEGQRLAGPTVL (SEQ ID NO: 1)
IELGITGPEGQRLAGPVL (SEQ ID NO: 2)
IELITGPEGQRLAGPTVL (SEQ ID NO: 3)
IELITGPEGQRLAGPVL (SEQ ID NO: 4)
EEIARARKSGQRVIGEPVAS (SEQ ID NO: 5),
For example, at least one of the partial sequences of SEQ ID NOs: 5 and 7,
Relates to a substantially pure hydantoinase containing at least one of the partial sequences, including at least one partial sequence having 60 to 100 percent identity, for example 100 percent.

Another embodiment of the present invention relates to a substantially pure hydantoinase, wherein the hydantoinase provides a crude extract of cellular material from an organism that naturally expresses the enzyme activity or is recombinantly expressed. As well as by subjecting the crude extract to the following continuous purification steps:
a) Ion exchange chromatography
b) Hydrophobic chromatography
c) Gel filtration
d) Affinity chromatography
e) Anion exchange
f) Gel filtration.

  Another embodiment of the invention relates to a substantially pure hydantoinase, wherein the hydantoinase comprises at least one of SEQ ID NOs: 1 to 5, and 7, and / or is isolated according to said method. Preferably, this hydantoinase is obtained from organisms of the genera Agrobacterium, Arthrobacter, Pseudomonas and Vigna, in particular Vigna angularis.

  Another embodiment relates to a hydantoinase, wherein the hydantoinase comprises at least one of SEQ ID NOs: 1 to 5, and 7, and in particular comprises substantially the purification steps in any order. Obtained from Azuki bean (Vigna angularis) and isolated according to the method described above, showing protein bands at 55 ± 10, 55 ± 5 or 55 ± 2, or about 55 kD, especially after SDS-PAGE under reducing conditions .

  Another aspect of the present invention relates to the use of said hydantoinase for the process of producing β-amino acid precursors or β-amino acids with a biocatalyst according to the present invention.

  Specifically, the hydantoinase is an organism of the genus Agrobacterium, Arthrobacter, Pseudomonas and Vigna, particularly hydantoinase derived from Vigna angularis. be able to.

  Another aspect of the present invention relates to the use of any of the above hydantoinases for the method of the present invention.

In the following, the present invention will be described in more detail and reference may be made to the figures here.
1 shows an SDS-PAGE gel of hydantoinase purified from azuki bean (Vigna angularis). Two example methods for synthesizing racemic 6-substituted dihydrouracil are shown. The separation of N-carbamoyl-β-phenylalanine (NCBPA), 6-phenyldihydrouracil (6-PDHU), and enzyme by anion exchange chromatography (DEAE column) is shown. The results of reverse phase chiral HPLC after hydantoinase catalyzes the hydrolysis of 6-PDHU are shown. The conversion ratio of 6-PDHU to the N-carbamoyl derivative (NCBPA) with time and the excess ratio of each enantiomer are shown. Figure 2 shows cyclization of N-carbamoyl-β-phenylalanine in the presence or absence of hydantoinase. Figure 2 shows the pH dependence of chemical hydrolysis of 6-DPHU in Tris buffer without hydantoinase. The pH dependence of the enzymatic conversion of 6-DPHU by hydantoinase is shown. Figure 3 shows hydantoinase conversion over time for various pH value / buffer combinations. The reaction catalyzed by D-hydantoinase (Sigma) from azuki bean (Vigna angularis) is shown. The influence of the organic cosolvent on the conversion of 6-PDHU catalyzed by hydantoinase is shown. Figure 2 shows the effect of various hydantoinase concentrations on product formation and enantiomeric excess. The production of NCBPA in a 25 mL reaction is shown. The enantiomeric excess of S-NCBPA in the 25 mL reaction solution is shown.

(Detailed description of the invention)
A. Definitions In the context of the present invention, it catalyzes the hydrolysis of substituted hydantoins and / or substituted dihydropyrimidine rings, such as “hydantoin and / or dihydropyrimidine rings such as 5-phenylhydantoin and 6-phenyldihydrouracil. The “enzyme” must exhibit the ability to catalyze the hydrolysis of at least one compound of formula (I) to form a compound of formula (II). Specifically, such an enzyme may be referred to as a hydantoinase and / or dihydropyrimidinase, but an enzyme whose natural substrate is a hydantoin ring is preferably referred to as a hydantoinase, and the natural substrate is a dihydrouracil ring. The enzyme will preferably be referred to as dihydropyrimidinase.

In the context of the present invention, dihydropyrimidinase has the ability to hydrolyze the dihydropyrimidine ring, in addition to the ability to hydrolyze hydantoin (as shown in Formula IV), as shown in Scheme 3 (± ) -5-characterized as an enzyme with the ability to resolve mono-substituted hydantoins.

Scheme 3

On the other hand, hydantoinases generally show little hydrolytic activity on dihydropyrimidines, but can hydrolyze hydantoins and (±) -5-monosubstituted hydantoins. An assay suitable for hydrolyzing hydantoin or dihydropyrimidine, and therefore suitable for further elucidating enzymes applicable to the process of the present invention, is described, for example, in Example 1.

  The term "about" can vary ± 25% about the indicated value, specifically ± 15%, ± 10%, ± 5% or ± 4% or ± 3% or ± 2% or ± 1 % Indicates the possibility of fluctuation.

In the context of the present invention, the enantioselectivity is that the enantiomeric excess “ee” of the S enantiomer is at least 50%, preferably at least 80%, more preferably at least 90%, specifically above 93%. , Especially at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, wherein the enantiomeric excess is expressed in percent (%). And calculated as follows based on the respective concentrations of the S and R enantiomers:
ee (%) = [(S enantiomer-R enantiomer) / (S enantiomer + R enantiomer)] x 100.

  As used herein, “substantially pure” protein or enzyme means that the desired purified protein is demonstrated in a single band after polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). , Essentially free of contaminating cell components (eg 90, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% (by dry weight) corresponds to the protein) . Furthermore, the expression “substantially pure” describes a molecule that is uniform based on one or more purity or uniformity characteristics used by those skilled in the art. For example, a substantially pure protein is one that exhibits constant and reproducible properties within standard experimental deviations for the following parameters: molecular weight, chromatographic transfer, amino acid composition, amino acid Sequence, N-terminal blocked or unblocked, HPLC elution profile, biological activity, and other parameters. However, this expression does not exclude artificial or synthetic mixtures of the protein with other compounds. In addition, this expression does not exclude fusion proteins of said protein that are optionally isolated from recombinant hosts.

B. Substrate for the method of the invention Residues R ¹ and / or R ² of the substrate of general formula (I) can be selected from linear or branched lower alkyl groups, which can be It may be substituted at multiple places. Residues R ¹ and R ² are specifically straight-chain or branched alkyl groups containing 1 to 10 carbon atoms, in particular 2 to 6 carbon atoms, such as methyl, ethyl, i- or n-propyl. A group consisting of a group, sec- or tert-butyl group, n-pentyl group, 2-methylbutyl group, n-hexyl group, heptyl group, octyl group, nonyl group, decyl group. Examples of branched alkyl groups include isopropyl, isobutyl, isopentyl, 2,2-dimethylpropyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, isoheptyl, 2-ethylbutyl, 3- Methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2,3-trimethylbutyl, isooctyl, 3-methylheptyl, 4-methylheptyl, 2,2-dimethyl There are hexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2,3-trimethylpentyl, 2,2,4-trimethylpentyl, 2,2,5-trimethylpentyl, and isononyl groups.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from linear or branched lower alkenyl groups, which may be substituted in one or more places . An example is an unsaturated analog of a lower alkyl group, which contains one or more carbon-carbon double bonds at any possible position of the carbon chain. Examples include vinyl group, allyl group (2-propenyl), but-1-enyl group, cis-but-2-enyl group, trans-but-2-enyl group, penta-1-enyl group, cis- Penta-2-enyl group, trans-pent-2-enyl group, 2-methylbut-1-enyl group, 2-methylbut-2-enyl group, 3-methylbut-1-enyl group, hexa-1-enyl group, Hex-2-enyl group (both cis-isomer and trans-isomer), hexa-3-enyl group, 2-methylpent-1-enyl group, 2-methylpent-2-enyl group, 3-methylpent-2-enyl Group, 4-methylpent-1-enyl group, 4-methylpent-2-enyl group, 2-ethylbut-1-enyl group, 2,3-dimethylbut-1-enyl group, 2,3-dimethylbut-2-yl group An enyl group and a 3,3-dimethylbut-1-enyl group.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from cyclic alkyl groups, which are compounds having a cyclic carbon skeleton, in particular 3 to 10 carbon atoms A compound having a cyclic carbon skeleton consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclononyl. In addition, monounsaturated or polyunsaturated analogs of cyclic alkyl groups such as cyclobutenyl group, cyclopentenyl group, cyclopentadienyl group, cyclohexenyl group, cycloheptenyl group, cyclohexadienyl group, and cycloheptadienyl group are included. It is. A cyclic alkyl group, or a monounsaturated or polyunsaturated analog thereof, may be substituted at one or more positions. Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from monocyclic or polycyclic aryl groups, which may be substituted at one or more positions. Good. Examples of aryl groups include monocyclic or polycyclic (especially bicyclic) aromatic groups, specifically phenyl or naphthyl groups attached via any ring carbon atom, such as 1- There are naphthyl and 2-naphthyl.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from monocyclic or polycyclic heteroaryl groups, which are substituted in one or more places. Also good. For example, heteroaryl groups are from pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, tetrahydropyran, tetrahydrothiopyran, pyrinide, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thioazole, pyran, pyrimidine, pyridazine, and pyrazine. Can be guided. Examples of bicyclic groups are condensates of said aryl or heteroaryl groups or their respective derivatives with additional aryl groups, heteroaryl groups or their derivatives, such as coumarol, indole, quinoline, purine and It is a group derived from naphthalene. The monocyclic or polycyclic heteroaryl group may be substituted at one or more positions, and is attached to each ring atom of the general formula (I) via any ring member atom, preferably a ring member carbon atom. Can be combined. Examples of suitable groups include 2-thienyl group, 3-thienyl group, 2-furanyl group, 3-furanyl group, 2-pyridyl group, 3-pyridyl group, 4-pyridyl group, 2-thiazolyl group, 4- Thiazolyl group, 5-thiazolyl group, 4-methyl-2-thienyl group, 3-ethyl-2-thienyl group, 2-methyl-3-thienyl group, 4-propyl-3-thienyl group, 5-n-butyl- 2-thienyl group, 4-methyl-3-thienyl group, 3-methyl-2-thienyl group, 3-chloro-2-thienyl group, 4-bromo-3-thienyl group, 2-iodo-3-thienyl group, There are 5-iodo-3-thienyl groups, 4-fluoro-2-thienyl groups, 2-bromo-3-thienyl groups, and 4-chloro-2-thienyl groups.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from linear or branched alkoxy groups, which may be substituted at one or more sites. In this case, the compound of the general formula (I) is linked to a group according to any of the above definitions via an oxygen atom, which group may be, for example, a linear or branched, substituted Lower alkyl group; linear or branched lower alkenyl group which may be substituted; cyclic alkyl group which may be substituted; monocyclic or polycyclic aryl group which may be substituted (aryl Also referred to as oxy groups); or monocyclic or polycyclic, optionally substituted heteroaryl groups. Examples of alkoxy groups are methoxy groups (—O—CH ₃ ), ethoxy groups (—OC ₂ H ₅ ) and groups of the general formula —OC _n H _{2n + 1} , where n is preferably 1 to 10 It is an integer up to.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from amino groups and linear or branched alkylamino groups, which are substituted in one or more places. Also good. In this case, the compound of general formula (I) is linked via a nitrogen atom, which itself has two hydrogen atoms (primary amine, amino group), one hydrogen and one organic residue ( Is bound to a secondary amine, —NHR ¹ ), or two organic residues (tertiary amine, —NR ¹ R ² ), which organic residues R ¹ and R ² are independent of each other, as defined above Arbitrary group, for example, linear or branched lower alkyl group which may be substituted; linear or branched lower alkyl group which may be substituted; cyclic alkyl group which may be substituted; Monocyclic or polycyclic, optionally substituted aryl groups; and monocyclic or polycyclic, optionally substituted heteroaryl groups can be selected. An example of an amino group is a primary amino group (—NH ₂ ). Examples of secondary amino groups are methylamino groups (—NH—CH ₃ ), ethylamino groups (—NH—C ₂ H ₅ ), and groups of the general formula —NH—C _n H _{2n + 1} , This n is preferably an integer from 1 to 10. Examples of tertiary amino group, dimethylamino group _{(-N (CH 3) 2)} , diethylamino group _{(-N (CH 2 CH 3)} 2), dipropylamino group _{(-N (CH 2 CH 2 CH} 3) ₂ ), a dibutylamino group (—N (CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ), a methylethylamino group, a methylpropylamino group, and the like.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from linear or branched alkylthio groups, which may be substituted at one or more positions. In this case, the compound of the general formula (I) is linked via a sulfur atom to a group according to any of the above definitions, such as a straight-chain or branched lower alkyl optionally substituted A linear or branched lower alkenyl group which may be substituted; a cyclic alkyl group which may be substituted; a monocyclic or polycyclic aryl group which may be substituted (also referred to as a thioaryl group) Or a monocyclic or polycyclic, optionally substituted heteroaryl group. Examples of alkylthio groups are thiomethyl (-S-CH ₃ ), thioethyl (-SC ₂ H ₅ ) and groups of the general formula -SC _n H _{2n + 1} , where n is preferably 1 to 10 It is an integer up to.

Residues R ¹ and / or R ² of the substrate of general formula (I) can be further selected from linear or branched acyl groups, which may be substituted at one or more sites. Acyl groups have the general formula —CC (O) —R, which can be an alkyl group, an aryl group, or a heteroaryl group as described herein. When R is hydrogen or OH, -C (O) -R becomes an aldehyde group or a carboxyl group, respectively.

The above groups may be appropriately substituted at one place or a plurality of places. A hydrogen atom attached to a carbon atom is, for example, independently of one another, another carbon compound, such as a linear or branched lower alkyl or alkenyl group, a cyclic alkyl group, an aryl or heteroaryl group; a halogen such as fluorine, chlorine, Bromine and iodine; or optionally substituted with heteroatoms or heteroatom-containing compounds. Examples of heteroatom-containing compounds are —OH, —SH, —NO ₂ , —NO ₃ , —NH ₂ , —SO ₃ , —SO ₄ groups, alkoxy groups and NR ³ R ⁴ , where R ³ And R ⁴ each independently represents H, a methyl group or an ethyl group.

In certain embodiments of the methods of the invention, R ² is H. In a further preferred embodiment, R ² is H and R ¹ is concomitantly selected from H, a methyl group, an optionally substituted aryl group, in particular an optionally substituted monocyclic aryl group. Preferred examples are a phenyl group, a mono-substituted or a poly-substituted phenyl group, and the substituents are independently of each other, for example, halogen, methyl, ethyl, —OH, —NH 2, —NO ² and —SO ³ H You can choose from. Substituted phenyl groups include, for example, 2-FC ₆ H ₄ , 3-FC ₆ H ₄ , 4-FC ₆ H ₄ , 2-Cl-C ₆ H ₄ , 3-Cl-C ₆ H ₄ , 4- Cl-C ₆ H ₄ , 2,3-FC ₆ H ₃ , 2,4-FC ₆ H ₃ , 2,5-FC ₆ H ₃ , 2,3-Cl-C ₆ H ₃ , 2,4-Cl -C ₆ H ₃ , 2,5-Cl-C ₆ H _{3 and the} like.

In a more specific embodiment, R ² is H and R ¹ is H, —CH ₃ , —CH ₂ CH ₂ CH ₂ NH—C (NH) NH ₂ , —CH ₂ CONH ₂ , —CH ₂ COOH. , -CH ₂ SH, -CH ₂ CH ₂ CONH ₂ , -CH ₂ CH ₂ COOH, -CH ₂ (C ₃ H ₃ N ₂ ), where C ₃ H ₃ N ₂ represents an imidazole group, -CH (CH _{3) CH 2 CH 3, -CH} 2 CH (CH 3) 2, -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 SCH 3, -CH 2 (C 6 H 5), - CH 2 OH, -CH (OH) CH ₃ , -CH ₂ (C ₈ H ₆ N), where C ₈ H ₆ N represents an indole residue, -CH ₂ (C ₆ H ₄ ) OH, -CH (CH ₃ ) ₂ , -CH ₂ -SeH, -CH ₂ -CH ₂ -SeCH ₃ (selenomethionine),-(C ₆ H ₅ ), pFC ₆ H ₄ , and p-Cl-C ₆ H ₄ Is done.

C. Process Conditions At least one enzyme, selected from hydantoinase and dihydropyrimidinase, present in the process for producing β-amino acid precursors naturally produces the enzyme (s), or Recombinantly produced, may be present in live cells, collected cells, dead cells, permeabilized cells, crude cell extracts, purified extracts, or in essentially pure form, Alternatively, it can exist in a completely pure form. The at least one enzyme may be present in a solution or may be an enzyme immobilized on a carrier. One or more enzymes may exist simultaneously as soluble and immobilized forms.

  The process of the invention can be carried out in various conventional scales known to those skilled in the art, for example from laboratory scale (reaction volumes from several milliliters to tens of liters) to industrial scale (several liters to thousands of cubic meters) Up to (reaction volume). Use chemical reactors if hydantoinase and / or dihydropyrimidinase are used in encapsulated by non-viable permeabilized cells, in the form of nearly purified cell extracts, or in purified form be able to. A chemical reactor typically allows for control of the amount of at least one enzyme, the amount of at least one substrate, the pH, temperature, and circulation of the reaction. If at least one enzyme is present in a living cell, the process is fermentation. In this case, biocatalytic production takes place in a bioreactor (fermentor), but the parameters necessary for the living conditions suitable for living cells (eg nutrient-containing medium, temperature, aeration, oxygen or other The presence or absence of gas, antibiotics, etc.) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, for example, to optimize chemical or biotechnological methods from laboratory scale to industrial scale, or to optimize process parameters. Familiar with the procedure, these are also widely described in the literature (for biotechnological methods, see, for example, Crueger und Crueger, Biotechnologie-Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munchen , Wien, 1984).

  Cells containing at least one hydantoinase and / or dihydropyrimidinase can be obtained by physical or mechanical means such as ultrasound or radio frequency pulses, French press; or chemical means such as hypotonic media, lytic enzymes, And the surfactant present in the medium; or a combination of such techniques. Examples of surfactants are digitonin, n-dodecyl maltoside, octyl glycoside, Triton® X-100, Tween® 20, deoxycholic acid, CHAPS (3-[(3-cholamidopropyl) Dimethylammonio] -1-propanesulfonic acid), Nonidet® P40 (ethylphenol poly (ethylene glycol ether)), and the like.

  If at least one enzyme is immobilized, it is attached to an inert carrier. Suitable carrier materials are known in the art and are described, for example, in EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773, and references cited therein (they are All are included herein, especially for carrier materials). Examples of suitable support materials are clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powder, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenol formaldehyde resins Polyurethanes and polyolefins such as polyethylene and polypropylene. In order to prepare an enzyme bound to a carrier, the carrier material is usually used in the form of a fine powder, but a porous material is preferred. The particle size of the carrier material usually does not exceed 5 mm, in particular 2 mm or less. If at least one enzyme is present in the whole cell preparation, the whole cell preparation may be present in a free state or in an immobilized form. Suitable carrier materials are, for example, calcium alginate or carrageenan. Both enzymes and cells can be linked directly by glutaraldehyde. Various immobilization methods are known in the art (eg, J. Lalonde and A. Margolin “Immobilization of Enzymes” in K. Drauz und H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991 -1032, Wiley-VCH, Weinheim).

D. Enzyme β-amino acid precursor is produced by biocatalyst, in particular at least one enzyme used in the process for enantioselective production of β-amino acid precursor by biocatalyst is from hydantoinase and dihydropyrimidinase Preferably, it is selected from any hydantoinase or any dihydropyrimidinase obtained from azuki bean (Vigna angularis). During biocatalytic production of β-amino acid precursors, one or more hydantoinases, one or more dihydropyrimidinases, or any combination thereof may be present. Preferably at least one hydantoinase is present. The at least one enzyme is preferably an enantioselective enzyme, with D-dihydropyrimidinase and / or D-hydantoinase being preferred. In particular, the enzyme is D-hydantoinase. At least one enzyme has the ability to generate the corresponding (S) -N-carbamoyl-β amino acid by hydrolyzing (±) -6-monosubstituted dihydrouracil, eg (±) -6-phenyldihydrouracil Preferably have the ability to produce (S) -N-carbamoyl-β-phenylalanine.

If this at least one enzyme is to be purified, general methods known to those skilled in the art can be used. After disrupting the cells, the crude extract can be obtained by centrifugation or filtration, specifically to separate proteins from cell debris, membrane fragments or cell ghosts. Additional or alternative purification steps include gel filtration, ion exchange chromatography (eg with Q-Sepharose), hydrophobic chromatography, reverse phase chromatography, ultrafiltration, crystallization, salting out, dialysis, Examples include native gel electrophoresis, immunoprecipitation, affinity chromatography. Suitable methods are described, for example, in Cooper, FG, Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York, or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin. According to a preferred embodiment, the at least one enzyme is present in at least partially purified form or essentially purified form. In the context of the present invention, other proteins normally present in the cell or organism used as the enzyme source must be clearly not detected by Coomassie staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme is basically in a purified form, but after silver staining, it may still be identified as a band on the SDS-PAGE gel. If other protein bands are indistinguishable after SDS-PAGE or only faintly discernible, the enzyme is present in a completely pure form. Isolation of hydantoinases has been described in the literature (eg Morin, Enzyme Microb. Technol. 15: 208-214, 1993; Fan and Lee, Biochemical Engineering J. 8: 157-164, 2001). Preferred extraction methods include at least 3, in particular at least 4, at least 5, or all of the following steps, preferably in the following order:
a) Ion exchange chromatography (eg using a Q-Sepharose FF-chromatography column)
b) Hydrophobic chromatography (eg using a phenyl sepharose column)
c) Gel filtration (eg using a Superdex Prep Grade 200 gel filtration column)
d) Affinity chromatography (eg using Blue HiTrap 5 ml affinity chromatography column)
e) anion exchange chromatography (eg using a Mono Q HR 5/5 anion exchange column) and
f) Gel filtration (eg using a Superose 6 prep grade gel filtration column).

  Suitable sources for at least one hydantoinase and / or dihydropyrimidinase are microorganisms and higher organisms such as bacteria and archaea, yeasts, fungi, plants and animals. Examples of bacteria include Acidovorax (e.g. A. avenae); Agrobacterium (e.g. A. radiobacter, A. tumefaciens, A. sp IP I-671, A. tumefaciens RU-OR, and A. tumefaciens NRRL B 11291); Arthrobacter ( A. crystallopoietes AM2), Aurantimonas (e.g. A. sp. SI85-9A1); Brucella (e.g. B. canis, B. ceti, B. ovis, B. pinnipedialis, B. suis); Burckholderia (e.g. B. sp. H160, B. phymatum, B. phytofirmans); Dickeya (e.g. D. dadantii); Jannaschia (e.g. J. sp. CCS1); Mesorhizobium (e.g. M. loti); Oceanicola (e.g. O. batsensis); Ochrobactrum (e.g. O. intermedium, O. sp. G21, O. anthropi); Polaromonas (eg P. naphthalenivorans), Reinekea (eg R. sp. MED297); Rhizobium (eg R. leguminosarum); Verminephrobacter (eg V. eiseniae); Rhodobacter (eg R. sphaeroides); Vibrio (eg V. cholerae); Fulvimarina (eg F. pelagi). An example of a simple metazoan organism is Trichoplax adherens, and examples of lower and higher plants are Arabidopsis (eg, A. thaliana); soybean rhizobia Genus (Bradyrhizobium) (eg B. sp. ORS278; B. japonicum, B. sp. BTAi1); Chlamydomonas (eg C. reinhardtii); Soybean genus ((Glycine) (Glycine max )); Medicago (M. trunculata); Oryza (eg, O. sativa; Physcomitrella (eg, P. patens)); P. sitchensis); Populus (eg P. trichocarpa); Ricinus (eg R. communis); Sorghum (Sorghum) For example, S. bicolor), Vitis (for example, V. vinifera), Zea (for example, corn), etc. Examples of animals include the genus Galus (for example, Sekishokukei ( G. gallus); human (Homo sapiens); macaca (eg, Rhesus monkey (M. mulatta); chimpanzee (P. troglodytes), and Xenopus (eg, Africa) Xenopus (X. laevis), X. tropicalis).

  Particularly preferred sources of at least one hydantoinase and / or dihydropyrimidinase are cowpea (especially Vigna angularis), Agrobacterium, Arthrobacter and Pseudomonas (Pseudomonas) ( In particular, according to the latest taxonomy, it is Burkholderia. Azuki bean hydantoinase is commercially available from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA).

Particularly preferred are hydantoinases and / or dihydropyrimidinases containing at least one partial sequence with 60 to 100 percent identity to at least one of the following partial sequences:
IELGITGPEGQRLAGPTVL (SEQ ID NO: 1)
IELGITGPEGQRLAGPVL (SEQ ID NO: 2)
IELITGPEGQRLAGPTVL (SEQ ID NO: 3)
IELITGPEGQRLAGPVL (SEQ ID NO: 4)
EEIARARKSGQRVIGEPVAS (SEQ ID NO: 5)
ITGPEGQRLAGP (SEQ ID NO: 7).

  The degree of identity for any of SEQ ID NOs: 1-5 or 7 can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. . In particular, the degree of identity for any of SEQ ID NOs: 1-5 or 7 is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% Or at least 99%. Among them, any of SEQ ID NOs: 1-5 or 7 can be identical (100% identical) to the sequences contained in hydantoinase and / or dihydropyrimidinase. The hydantoinase and / or dihydropyrimidinase properties of any given protein can be readily measured by the hydantoinase and / or dihydropyrimidinase assays described herein.

  Such hydantoinase and / or dihydropyrimidinase in addition to or in place of hydantoinase and / or dihydropyrimidinase present in or extracted from an organism or microorganism Functionally equivalent ones can be used.

A functional equivalent is a variant that differs from the natural amino acid sequence in at least one place, but still at least partially retains the enzymatic activity of hydantoinase and / or dihydropyrimidinase. A functional equivalent is an addition, substitution, deletion of one or more, eg 1 to 20, 1 to 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids And / or may include inversions, but this may occur at any sequence position as long as the equivalent does not completely abolish catalytic activity. A functional equivalent may also be an enzyme with altered substrate and / or product turnover rates and / or altered selectivity of a particular substrate (eg, larger or more hydrophobic) , Functional equivalents that accept substrates having R ¹ and / or R ² groups). Non-limiting examples of substitutions that are likely to at least partially preserve enzyme activity are described below:

  Functional equivalents further include mature hydantoinase and / or precursors of dihydropyrimidinase protein, and salts thereof. The salt includes a salt of a carboxyl group and an acid addition salt of an amino group. Salts of carboxyl groups can be made by methods known in the art, including salts of inorganic salts such as sodium, calcium, ammonia, iron and zinc, and organic bases such as triethanol. There are salts formed with amines such as amines, arginine, lysine, piperidine and the like. Acid addition salts include salts with inorganic acids such as hydrochloric acid or sulfuric acid, and salts formed with organic acids such as acetic acid and oxalic acid.

  Functional equivalents can also be obtained by modifying naturally occurring hydantoinases and / or dihydropyrimidinases at one or more amino acid side chains, or at the N-terminus or C-terminus. Such derivatives include, for example, aliphatic esters of carboxyl groups, amides of carboxyl groups (obtained by reaction with ammonia or primary or secondary amines), N-acyl derivatives of free amine groups (obtained by reaction with acyl groups). ), Or O-acyl derivatives of free hydroxy groups (obtained by reaction with acyl groups). Also included are functional equivalents obtained by glycosylation patterns that differ from the natural glycosylation pattern (eg, after artificial glycosylation or deglycosylation).

  Functional equivalents also include enzymes obtained from other organisms or microorganisms, as well as naturally occurring variants of related enzymes.

  Functional equivalents also include single domains or sequence motifs with the desired activity of hydantoinase and / or dihydropyrimidinase.

  In addition, a functional equivalent is a fusion protein comprising a naturally occurring sequence or functional equivalent thereof combined with at least one functionally different sequence, linked at the N-terminus or C-terminus. There may be, but this does not abolish the hydantoinase and / or dihydropyrimidinase activity of the protein. Examples of such functionally distinct sequences are signal peptides (eg, directing the excretion of the fusion protein), enzymes (eg, to add other enzymatic activities), or portions of immunoglobulin (eg, fusion proteins). To immobilize.

  Functional equivalents also include homologues of naturally occurring proteins. Said homologue is at least 60% homologous to a naturally occurring amino acid sequence, preferably at least 75%, and in particular at least 85%, such as 90%, 95% or 99% homology (Pearson and Lipman , Proc. Natl. Acad, Sci. (USA) 85 (8), 1988, 2444-2448). The “% homology” of a given polypeptide specifically represents the percentage identity of amino acid residues with respect to the full length of the reference enzyme or enzyme subunit.

  Functional equivalents can also be made by methods known in the art. Methods for modifying genes and, as a result, methods aimed at proteins encoded by such genes have been known for some time, for example, one or more nucleotides of a gene are specifically exchanged, Site-directed mutagenesis (Trower MK (Hrsg.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey), saturation mutagenesis where codons for any amino acid can be exchanged or added at any position in the gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22: 1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22: 541; Barik S (1995) Mol Biotechnol 3 : 1) Error-prone polymerase chain reaction (error-prone PCR) (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18) : 3739); For example, an increase in the mutation rate is observed due to the defective DNA repair mechanism (Gerer et al., Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Hrsg.) In vitro mutagenesis protocols. Humana Press, New Jersey), or fragments obtained after first creating and digesting a pool of closely related genes DNA shuffling (Stemmer WPC (1994) Nature 370: 389; Stemmer WPC (1994) Proc Natl Acad Sci USA). 91: 10747).

  Depending on the method used, one skilled in the art will introduce a gene bank after introducing purely random mutations, or more directed mutations into genes or non-coding regions (which may be related to expression control). Can be produced. The required methods are known to those skilled in the art and are described, for example, in Sambrook and Russell, Molecular Cloning. 3. Edition, Cold Spring Harbor Laboratory Press 2001.

  So-called “directed evolution” methods (especially Reetz MT und Jaeger KE (1999), Topics Curr Chem 200: 31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial enzymes by directed evolution : Demain AL, Davies JE (Hrsg.) Manual of industrial microbiology and biotechnology. (As described in the American Society for Microbiology), in a directed approach (as opposed to completely random) Moreover, functional equivalents can be produced on a large scale. In the first stage, a gene bank for each protein (eg hydantoinase and / or dihydropyrimidinase) is created, for example by applying the method. The gene bank is expressed in a suitable manner, for example by bacteria or phage display systems. In addition, methods for controlling protein localization in the host by selecting appropriate expression vectors are known in the art. The protein may be localized in the cytoplasm within the cell or within the membrane by the addition of a membrane anchor, but is localized extracellularly by the addition of a signal peptide containing a signal peptidase recognition sequence Sometimes. In the next second step, clones expressing proteins with the desired properties are selected or screened. When using selection methods, clones that express proteins with desirable properties will survive because such proteins promote or guarantee the survival of host cells (eg, at the use of specific substrates or at specific temperatures). Enzyme that allows the growth of). When using the screening method, all clones survive. Clones expressing proteins with the desired properties are identified by suitable assays that can be readily devised by those skilled in the art. For example, when searching for proteins with specific binding properties, host cells that adhere to a surface coated with a substrate appropriate for the desired binding ability can be screened. Host cells that express such proteins can attach to the substrate via the expressed protein, but host cells that express nonfunctional proteins cannot. When searching for functional equivalents having specific catalytic properties, host cells can be cultured in substrate-containing media or substrate-containing agar plates. The presence of functional equivalents is indicated, for example, by a color change after substrate modification by such functional equivalents (as necessary to allow contact between the functional equivalent and the substrate-containing medium). After lysing the host cells). Functional equivalents of hydantoinase or dihydropyrimidinase can be screened on media containing dihydrouracil with the desired substitution.

  In this context, use automated systems (eg pipetting robots for microtiter plates, screening robots, image processing systems to identify colonies on agar plates) to enable high-throughput screening. You can also. Each gene of the host cell that expresses a functional equivalent, most or at least partially having characteristics corresponding to the desired properties, is subjected to one or more mutations. Mutation and selection or screening steps can be repeated in an iterative process until the functional equivalent has satisfactory desirable properties.

  Loss of function by inserting a large number of mutations into a protein sequence rather than improving certain properties (eg enzyme turnover) or gaining new properties (accepting new substrate classes) Although more likely, it is possible to screen for proteins with desirable properties in an oriented manner by applying an iterative approach. When performing an iterative approach, gene libraries with mutations are created based on the nucleotide sequence of a reference protein (eg, individual hydantoinase or dihydropyrimidinase) such as a wild-type protein. In this context, the mutation rate at the selected nucleotide level causes a relatively small number of amino acids, for example 1 to 3 amino acids, to mutate in the corresponding translated peptide or protein. The resulting peptide or protein is then subjected to desirable characteristics (eg, increased catalytic activity for one or more substrates, expanded or altered substrate range, elevated or altered temperature or pH value, or a specific solvent For increased stability in the presence). Based on the sequence of the protein or peptide having these desirable properties (which may need only a few confirmations), a second gene library is created and again introduced with a low rate of mutations. The translated protein is then screened for the desired properties. The cycle of creating a mutated gene library and screening for peptides or proteins expressed based on that library can be repeated as many times as deemed necessary. Choosing a low mutation rate per cycle avoids situations where too many mutations accumulate and almost all proteins do not function. Low mutation rates (as well as concomitant low or slow expression of desirable properties) iteratively repeat mutations (which may result in functional equivalents) and selection (of desired functional equivalents) Which can lead to the accumulation of useful mutations and ultimately provide a high rate of successful proteins with desirable improved properties. Sequence analysis of functional equivalents with improved properties provides sequence information that identifies sequence positions or regions within the peptide or protein that are important for the desired properties. Examples for the production of proteins with desirable properties are described in Zhao und Arnold, Protein Engineering 12: 47-53 (1999), or May et al., Nature Biotechnology 18: 317-320 (2000). Other ways to implement will then become apparent.

  Functional equivalents can also be obtained by nucleic acid sequences derived from the nucleic acid sequence underlying the protein. Unless otherwise specified, the derived nucleic acid sequence has at least 80% identity to the original nucleic acid, or at least 90%, specifically about 91%, 92%, 93%, 94%, 95 Nucleic acids with%, 96%, 97%, 98% or 99% identity. While 100% identity means that the nucleotides of the two nucleic acid sequences are identical over the entire length of the nucleic acid, specifically, identity is determined by using the Vector NTI Suite 7.1 software from Infomax (USA) and the Clustal method. And determined by comparison of two nucleic acid sequences.

  Derived nucleic acids (single-stranded or double-stranded DNA or RNA sequences such as cDNA or mRNA) can be chemically synthesized from nucleotides as building blocks by methods known in the art. It can be chemically synthesized by condensing unit nucleotides or oligomers. For example, chemical synthesis can be performed by the phosphoramidite method ((Voet, Voet, 2. Ed., Wiley Press, New York, p. 896-897), annealing of synthetic oligonucleotides, and Klenow fragment of DNA polymerase. Gap filling, ligation reactions, and general cloning methods are known in the art and described, for example, in Sambrook et al., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, 1989. Yes.

  The derived nucleic acid can be identified in a database, but can also be isolated from a gene bank or organism. Specifically, the derived nucleic acid is obtained by using a nucleic acid corresponding to the peptide sequence described in SEQ ID NOs: 1-5 or 7 as a probe for hybridization or as a primer for polymerase chain reaction (PCR). It can be isolated. The probe or primer is usually at least 12, preferably at least 25, such as about 40 or about 75 contiguous sequences of sense or antisense strands of the nucleic acid sequence that may be derived from the original nucleic acid under stringent conditions. It contains a nucleotide sequence region that hybridizes with the selected nucleotide. Possible derived nucleic acid sequences include gene banks, cellular materials of the target organism, such as cells (preferably permeable cells) or cellular components extracted therefrom (preferably at least partially purified nucleic acids). Although it can be present in, this target organism is the organism to be examined for the presence of the derived nucleic acid. Cellular material can be separated on an agarose gel and applied to a membrane for Northern blot or Southern blot hybridization under standard conditions to detect the presence of nucleic acid fragments that bind to the probe used. Alternatively, it can be a template for a primer for a PCR reaction, which probe or primer is derived from a nucleic acid sequence based on any of the peptide sequences set forth in SEQ ID NOs: 1-5 or 7. Can be obtained. Isolate a hybridized nucleic acid band on a blot membrane or gel, or a template nucleic acid hybridized in a PCR reaction (which may have been amplified using forward or reverse primers derived from the original nucleic acid, or random primers) After cloning into an appropriate vector by methods known in the art (eg, Sambrook et al., 1989) or sequencing, or from a gene bank or cellular material, a longer, preferably full length gene. Can be used as a hybridization probe under more stringent conditions.

  Standard hybridization conditions vary for each nucleic acid, but are generally in aqueous buffer solutions from 0.1 x SSC to 5 x SSC (1 x SSC is 0.15 M NaCl, 14 mM sodium citrate , Corresponding to pH 7.2), or additionally in the presence of 50% formamide at a temperature of 42 ° C. to 58 ° C., for example 42 ° C. in 5 × SSC containing 50% formamide. Hybridization conditions for DNA: DNA hybrids preferably include 0.1 x SSC and a temperature of about 20 ° C to about 45 ° C, particularly 30 ° C to 45 ° C. Hybridization conditions for DNA: RNA hybrids preferably include 0.1 x SSC and a temperature of about 30 ° C to about 55 ° C, particularly 45 ° C to 55 ° C. The above temperature is an example of the hybridization temperature of a nucleic acid with a G + C content of 50% containing about 100 nucleotides in SSC buffer without formamide. Experimental conditions for DNA hybridization and other molecular biology techniques are described in molecular biology textbooks or experimental manuals (eg, Sambrook et al., 1989, Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1985; Hames and Higgins (eds.), Nucleic Acid Hybridisation: A Practical Approach, IRL Press at Oxford University Press, Oxford, 1985; Brown (ed.), Essential Moelcular Biolgoy : A Practical Approach, IRL Press at Oxford University Press, Oxford, 1991), but those skilled in the art know in the art, considering parameters such as nucleic acid length, hybrid type, and G + C content Can be calculated according to the formula An example of stringent conditions in Northern blots, for example, is the use of 0.1 x SSC containing 0.1% SDS (20 x SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0), and temperatures between 50 ° C and 70 ° C. Although included, 60 ° C. to 65 ° C. is preferred for eluting non-specifically bound probes or oligonucleotides. The selection and adaptation of stringent conditions is known in the art and is described in the literature (eg Sambrook et al., 1989).

  The derived nucleic acid can be isolated according to methods known in the art. An isolated nucleic acid molecule is to be understood as a nucleic acid molecule that is essentially separated from other nucleic acid molecules containing deviating sequences. Isolated nucleic acid molecules are preferably essentially free of cellular material or medium other than nucleic acid and, when made by chemical synthesis, are essentially free of chemical precursors or other chemicals. It is preferable.

  The derived nucleic acid can be appropriately cleaved with a restriction enzyme, ligated with a linker molecule, and then introduced into an expression construct. In the expression construct, the derived nucleic acid is operably linked to regulatory elements that control the expression of the corresponding polypeptide or protein sequence from the nucleic acid sequence, said nucleic acid sequence comprising a useful hydantoinase. Or a sequence encoding dihydropyrimidinase. The expression construct preferably contains a promoter upstream of the derived nucleic acid and a transcription termination sequence downstream thereof. Functional linkages are sequentially arranged such that the promoter, coding sequence, terminator, and optionally additional regulatory elements allow the proper functioning of each of the regulatory elements for expression of the coding sequence. Should be understood. Examples of elements to be operably linked include targeting sequences, enhancers, polyadenylation signals, and the like. Other useful elements include selectable markers, amplification signals, and origins of replication. Useful regulatory elements are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). In addition to or instead of the artificial regulatory elements or sequences, natural regulatory sequences may be present upstream of the hydantoinase or dihydropyrimidinase structural genes. Natural regulation can be switched off by genetic modification, or modified to reduce or increase expression. Similarly, genetic constructs may have a more simplified structure by preserving only natural regulatory elements without inserting artificial elements. Natural regulatory elements can be mutated so that regulation does not occur and gene expression is reduced or preferably increased. The hydantoinase or dihydropyrimidinase structural gene may be present as a single copy or multiple copies, but in some cases, as a mixed copy, ie, hydantoinase and dihydropyrimidinase may be present simultaneously in the gene construct.

  Examples of useful promoters are cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, lambda-PR- or lambda-PL- promoters (which are preferably used in gram-negative bacteria); and promoters amy and SPO2, or yeast promoters ADC1, MFalpha, AC, P -60, CYC1, GAPDH. Inducible promoters that allow induction of hydantoinase or dihydropyrimidinase expression after cell growth and accumulation of the appropriate cell density, such as temperature or compound-inducible promoters (eg, metallothionein promoter) may be used. Similarly, artificial promoters can be used.

The recombinant expression construct is preferably inserted into a suitable vector, such as a plasmid, which is then introduced into a suitable host cell. Vectors are known in the art and are described, for example, in Pouwels et al. (Eds.), Cloning Vectors, Elsevier, Amsterdam-New York-Oxford, 1985. In addition to plasmids, the vector may be any other vector known in the art, such as phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposon, IS element, fasmid, It can be cosmid and linear or circular DNA. Vectors can replicate autonomously in the host, but may be replicated chromosomally after being integrated into the chromosome. Examples of non-fusion protein expression vectors are pTrc (Amann et al., (1988) Gene 69: 301-315), and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Kalifornien (1990) 60-89). For example, examples of yeast expression vectors for expression in Saccharomyces cerevisiae are pYepSec1 (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan und Herskowitz (1982) Cell 30: 933- 943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for constructing useful vectors for fungi, particularly filamentous fungi, are described in van den Hondel, CAMJJ & Punt, PJ (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi. , JF Peberdy et al., Hrsg., S. 1-28, Cambridge University Press: Cambridge, examples of baculovirus vectors for expressing proteins in insect cells such as Sf9 cells include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and pVL series (Lucklow und Summers (1989) Virology 170: 31-39). Examples of mammalian cell vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195) Other expression systems for prokaryotic and eukaryotic cells are: Sambrook, Fritsch, and Maniatis, Molecular cloning:. A Laboratory Manual, 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Labor It is described in Chapters 16 and 17 of atory Press, Cold Spring Harbor, NY, 1989.

  The vectors can be used to create recombinant organisms that are transformed with at least one of those vectors and express hydantoinase or dihydropyrimidinase useful in the methods of the invention. The recombinant construct can be expressed after being introduced into a suitable host system. For introduction, methods known in the art such as coprecipitation, protoplast fusion, electroporation, chemical transformation, retroviral transformation and the like can be used. Suitable methods include, for example, Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, oder Sambrook et al. Molecular Cloning: A Laboratory Manual. 2. Aufl., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

  Suitable host organisms or host cells are organisms or cells that allow the expression of nucleic acids encoding hydantoinase or dihydropyrimidinase, or nucleic acids derived therefrom. Host cells include bacterial, fungal, yeast, plant or animal cells. Examples of bacteria are those of the genus Escherichia, such as Escherichia coli, Streptomyces, Bacillus, Pseudomonas or Burkholderia, which are eukaryotic microorganisms. Examples are the genus Saccharomyces cerevisiae, Aspergillus, and higher eukaryotic cells from animals or plants are, for example, Sf9-, CHO- or HEK293 cells, but higher true such as animals or plants. A single cell or aggregated cell of a nuclear organism is sometimes referred to herein as a microorganism.

  Selection of cells that have been successfully transformed can be performed based on the marker gene contained in the respective vector or expression cassette. Examples of marker genes include antibiotic resistance genes or genes for enzymes that catalyze the color reaction, which results in staining or fluorescence of the transformed cells. The cells can be selected by automated cell sorting. For example, successfully transformed cells carrying an antibiotic resistance gene for G418 or hygromycin can be selected on antibiotic-containing media or agar plates. Marker proteins expressed on the cell surface can be used for selection by affinity chromatography.

  The cells are cultured as described in section C above and can secrete hydantoinase or dihydropyrimidinase into the medium, so that the growth or survival of the cells as well as at least one substrate of general formula (I) Therefore, it is possible to produce a β-amino acid precursor from a biocatalyst. If hydantoinase or dihydropyrimidinase is not secreted into the medium, make cells permeable after growth and / or induction of hydantoinase or dihydropyrimidinase expression to cause biocatalytic production of β-amino acid precursors Can do. Alternatively, cells may be harvested to partially or completely purify hydantoinase or dihydropyrimidinase after growth and / or induction of hydantoinase or dihydropyrimidinase expression, resulting in a β-amino acid precursor Purified hydantoinase or dihydropyrimidinase can be used for the biocatalytic production of.

E. Additional process steps During the process for obtaining β-amino acid precursors or β-amino acids, one or more additional enzymes may optionally be present in the reaction. Such enzymes catalyze the conversion between the D- and L-enantiomers of the dihydrouracil substrate of general formula (I). Examples of suitable enzymes include dihydrouracil dehydrogenase (eg, of mammalian origin), dihydrouracil oxidase (Rhodotorula glutinis) and enoate reductase (formerly yellow enzyme).

A β-amino acid precursor of general formula (II) (β-amino acid carbamoyl derivative) is made by the method of the present invention, which is cleaved into the corresponding β-amino acid and N-carbamoyl moiety while remaining in the reaction mixture. can do. Alternatively, the beta amino acid precursor can be separated and cleaved from the reaction mixture containing the substrate of general formula (I) and at least one hydantoinase and / or dihydropyrimidinase to produce the beta amino acid. Cleavage can be done chemically or enzymatically. Chemical cleavage is carried out at an acidic pH, eg 1.0, with an equimolar amount of NaNO ₃ (β as described in Fan and Lee, Biochemical Engineering Journal 8: 157-164, 2001). Can be achieved by adding to the body. Acidic pH can be achieved, for example, with sulfuric acid. The cleavage reaction can be performed at low temperature, for example on ice. When the cleavage of the β-amino acid precursor of the general formula (II) is chemically achieved, it is preferable to carry out this reaction separately from the enzymatic reaction from the substrate of the general formula (I) to the β-amino acid precursor. Cleavage can be performed after enzymatic conversion is complete or after equilibrium is reached (temporal separation), but is performed in a separate reaction vessel after removal of the reaction medium containing the beta amino acid precursor. (Spatial separation). As an alternative to this batch method, continuous production can be achieved by contacting a reaction medium containing a substrate molecule of general formula (I) with an immobilized enzyme (hydantoinase and / or dihydropyrimidinase). However, the enzyme is thus retained in the reaction vessel, while the reaction medium that has exhausted the substrate can be separated for cleavage of the β-amino acid precursor and at the same time replaced with new reaction medium. Enzymatic cleavage can be performed by enzymes such as carbamoylase (eg EC3.5.1.77 or EC3.5.1.87) or β-ureidopropionase (EC3.5.1.6). Such enzymes can be obtained from commercial suppliers (eg, carbamoylase from Julich Chiral Solutions, Julich, Germany).

F. The pH value and buffer system The method of the invention for converting a substrate of general formula (I) to a β-amino acid precursor of general formula (II) is preferably carried out at a pH of about 7.0 to about 11.0 However, a pH of about 7.5 to 10.0 is preferred. Specifically, the pH is in the range of 7.5 to 8.5, and more particularly in the range of about 7.5 to 8.0. Particularly preferred pH values are about 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0.

  Any buffer suitable for said pH value or pH range can be used, such as phosphate buffer, borate buffer, and Tris buffer. Tris buffer is preferred in the pH range from 7.5 to 9.0, but phosphate buffer is useful in the pH range from 6.0 to 8.0. Particularly preferred is a reaction solution buffered with Tris. The concentration of the buffer is determined depending on the concentration of the substrate to be used, but is preferably in the range of 1 mM to 200 mM, for example, 1 mM to 100 mM, 1 mM to 50 mM. Specifically, the buffer concentration can be 2 mM to 25 mM, or 3 mM to 10 mM, for example 5 mM.

G. Temperature and duration of the reaction The process for the biocatalytic conversion of the substrate of general formula (I) can be carried out at any temperature that can withstand the hydantoinase and / or dihydropyrimidinase used. Usually, the temperature correlates with the optimal temperature for growth of the organism or microorganism, which encapsulates the enzyme or is used as the source of enzyme extraction, but can be easily determined by those skilled in the art. In general, the process can be carried out at temperatures between 30 ° C. and 60 ° C., in particular between 40 ° C. and 50 ° C. Examples of reaction temperatures are about 30 ° C, about 35 ° C, about 37 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, and about 60 ° C.

  The process can proceed until equilibrium is achieved between the substrate of general formula (I) and the beta amino acid precursor of general formula (II), but can also be stopped earlier. Typical process times are in the range of 1 minute to 25 hours, but in particular 10 minutes to 6 hours. Preferred reaction times are in the range from 1 hour to 4 hours, but in particular from 1.5 hours to 3.5 hours.

H. Additional components in the reaction medium Even if one or more organic cosolvents are included in the reaction medium in order to increase the solubility of the substrate with essentially hydrophobic R ¹ and / or R ² Good.

  Alternatively, the reaction can be carried out in a two-phase system consisting of an aqueous phase and a non-aqueous phase. Examples of suitable solvents for the non-aqueous phase are preferably aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; preferably 1 or Halogenated aliphatic hydrocarbons having 2 carbon atoms, such as dichloromethane, chloroform, tetrachloromethane, dichloroethane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; preferably 4 to 8 Aliphatic acyclic or cyclic ethers having carbon atoms such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, tetrahydrofuran; or esters such as ethyl acetate Acetate n- butyl; or ketones, for example methyl - iso-butyl ketone or dioxane, or mixtures thereof.

  In certain embodiments, the reaction medium is an aqueous reaction medium that contains an organic cosolvent, particularly as a single phase system. Examples of suitable cosolvents are butan-2-ol, methyl-tert-butyl ether (MTBE) and dimethyl sulfoxide (DMSO). When using MTBE, concentrations below saturation (about 12% by volume) in an aqueous medium are preferred, for example about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or 1% Is less than. MTBE can be used up to a saturating concentration in aqueous media (about 6% by volume), while DMSO basically increases the solubility of the substrate without interfering with the yield of the process. A concentration of up to about 2% by volume can be used.

I. Methods for isolating amino acids and N-carbamoyl amino acids from N-carbamoyl β-amino acids or β-amino acid recovery reaction media are known in the art, such as gel filtration, HPLC, reverse phase chromatography and ions Techniques such as exchange chromatography are included. Ion exchange chromatography, particularly anion exchange chromatography, is the preferred method. Suitable ion exchange matrices include strong ion exchangers (eg Q Sepharose based on quaternary ammonium) and weak anion exchangers (eg DEAE Sepharose based on diethylaminoethyl). Examples of preferred methods are anion exchange chromatography using a DEAE column, or extraction with an organic solvent, which uses ethyl acetate to extract the substrate from the biological conversion system, then the product. Use 2-butanol for extraction.

  The present invention will now be illustrated in detail by the following examples that illustrate the invention, but are not intended to limit the invention.

(Example)
Chemical experiments TLC was performed using plastic support plates coated with 0.2 mm thick silica gel 60F254 (Mackerey-Nagel). Plates were visualized by UV light (254 nm) or permanganate immersion. Chemicals were purchased from Sigma-Aldrich, Acros or Fluka unless otherwise indicated. Unless otherwise noted, all reagents are of standard laboratory grade, solvents are anhydrous and are used as supplied. 1H-NMR and 13C-NMR were recorded on a Bruker AC300 or AC400 spectrometer. The following abbreviations were used: δ, chemical shift; bs, broad singlet; d, doublet; dd, doublet of doublets; J, coupling constant; m, multiplet (Multiplet); q, quartet; qui, quintet; s, singlet; sep, septet. The chemical shift (δ) is recorded in parts per million (ppm) and the coupling constant (J) is recorded in Hz. The residual protic solvent, DMSO-d6 (δ H 2.50, qui) was used as an internal standard for the 1H NMR spectrum, and the 13C NMR shift was determined by using DMSO-d6 (δ C 39.5, sep) with a decoupled broad band. Used as standard.

Biotransformation experiments Unless otherwise stated, all experiments were performed in a 15 ml Falcon tube at a scale of 5 mM 6-PDHU (4.75 mg) in 5 mL Tris buffer (0.1 M pH 7.5) with 1 U of commercially available It was performed using hydantoinase derived from azuki bean (Vigna angularis) (35 mg, purchased from Sigma, St. Louis, USA) and incubated in a thermomixer (Falcon) at 750 rpm and a temperature of 50 ° C.

  Biotransformation was performed using “1 unit / reaction”, where 1 unit is defined as the amount that catalyzes the production of 1 μmol N-carbamoylglycine per minute from hydantoin.

Assay for hydrolysis of 5-substituted hydantoins or 6-phenyldihydrouracil To measure hydantoinase activity, D-in 5-phenylhydantoin (1.32 mg) dissolved in borate buffer (1.5 mL, 0.1 M, pH 9.0). Hydantoinase (10.5 mg) was added. Biotransformations were incubated in Eppendorf “Themomixer comfort”, (40 ° C., 1400 rpm). At defined time points, the reaction was terminated by taking an aliquot (100 μL) of the mixture and precipitating soluble proteins by denaturing the enzyme by addition of TCA (12% w / v, 175 μL). After centrifugation, the supernatant was collected and analyzed by HPLC, where the following parameters were used:
Column: Agilent Zorbax XBD-C18 4.6 mm x 50 mm, 3.5 μm.
Conditions: 25 ° C., H ₂ O: acetonitrile, 85:15 (+ 0.1% TFA) at 1 ml / min.

Retention time was 5-phenylhydantoin (3.6), N-carbamoyl α-phenylglycine (2.7). This assay step can be used to measure the hydantoinase activity of such enzymes by replacing D-hydantoinase with an enzyme that may have hydantoinase activity.

To measure dihydropyrimidinase activity, D-hydantoinase (10.5 mg) was added to 6-phenyldihydrouracil (1.14 mg) dissolved in Tris buffer (300 μL, 0.1 M, pH 7.5). Biotransformations were incubated in Eppendorf “Themomixer comfort”, (40 ° C., 1400 rpm). At defined time points, the reaction was terminated by taking an aliquot (100 μL) of the mixture, denaturing the enzyme (95 ° C., 5 min) and precipitating soluble protein with the addition of methanol (300 μL). After centrifugation, the supernatant was collected and analyzed by HPLC, where the following parameters were used:
c-18 rpHPLC: uniform concentration mobile phase, 25 ° C, H ₂ O: ACN, 90:10 + 0.1% TFA
Retention time: 6-phenyldihydrouracil (12.6), N-carbamoyl β-phenylalanine (NCBPA, 4, 9.6) TCA (4.3) DMSO (1.6)
Astec Chirobiotic T column, 25 cm x 4.6 mm, 5 μm: homogeneous concentration mobile phase, 5 ° C., 20 mM ammonium acetate (pH 6.5): MeOH, 70:30
Retention time: 6PDHU (15.9, 27.5), NCBPA (7.8, 6.8).

Isolation of hydantoinase from azuki bean (Vigna angularis) A protein having hydantoinase activity was isolated from azuki bean (Vigna angularis) as follows.

2.1 Bean extraction
885 g of azuki bean (purchased from a health food store) was pulverized with dry ice using an ordinary mixer in two divided amounts. To obtain azuki bean powder, add 4 L of extraction buffer (20 mM Tris, 10 mM ascorbic acid, 10 mM lysine, pH 7.5), stir the suspension at 4 ° C. overnight, filter through gauze and centrifuge separated. 2500 mL of crude extract with a protein content of 9.5 mg / ml was obtained and stored at −20 ° C. until the next chromatography step (using a chromatography column from Amersham Pharmacia or GE Healthare).

2.2 Ion exchange chromatography (Q-Sepharose Fast Flow)
A Q-Sepharose FF chromatography column (diameter 5 cm; length 21 cm) was washed with 200 mM Tris / HCL (pH 7.5), and running buffer A (20 mM Tris / HCl, 1 mM ascorbic acid, 1 mM L- The crude extract (5 g total protein) was loaded after equilibration with lysine hydrochloride, pH 7.5). A linear concentration gradient from buffer A to buffer B (20 mM Tris / HCl, 750 mM NaCl, 1 mM ascorbic acid, 1 mM L-lysine hydrochloride, pH 7.5) was applied to collect the active fraction.

  Ehrlich reagent was used to measure the activity of the fractions as follows: 50 mL protein sample was mixed with 50 μL 5-hydantoin (100 mM, 100 mM borate buffer, pH 9, medium) at room temperature for 10-60 Incubated for minutes (depending on enzyme content). Proteins present in the sample were completely precipitated by adding 150 μL of 12% trichloroacetic acid (TCA) and removed by centrifugation. 50 μL 4- (dimethylamino) -benzaldehyde (10% in 6 M HCl) was added to 200 μL supernatant and measured with a photometer at 450 nm.

2.3. Hydrophobic chromatography (Phenyl Sepharose Fast Flow)
A Phenyl Sepharose column with a diameter of 5 cm and a length of 21 cm was used. Add water and dilute the pooled ion exchange chromatography active fraction (280 ml) to 500 ml and add 67 g (NH ₄ ) ₂ SO ₄ to 25% saturation, resulting mixture Was applied to the column. Running buffer A (25 mM saturated with 20 mM Tris, (NH ₄ ) ₂ SO ₄ , 1 mM lysine, 1 mM ascorbic acid, pH 7.5; also used as washing buffer) was added at a low flow rate of 15 mL / min. The column environment was linearly changed to running buffer B by adding 2 times the column volume of running buffer B (20 mM Tris, 1 mM lysine, 1 mM ascorbic acid, pH 7.5). Double volume of buffer B was added for elution. Thereafter, buffer C (10 mM Tris / HCl, pH 7.5, 10% 2-propanol) was used for washing. The active fractions showing the maximum absorption as measured by the hydantoinase assay using Ehrlich reagent were pooled.

2.4 Molecular sieve chromatography (Superdex Prep Grade 200 gel filtration)
For the next purification step, a Superdex 200 column (diameter 2.6 cm, length 60 cm) was run at a flow rate of 4 mL / min. To the pooled active fraction obtained from hydrophobic chromatography, 70.2 g (NH ₄ ) ₂ SO ₄ was added to 80% saturation and the resulting mixture was centrifuged (20 min, 12,000 rpm) To obtain pellets. The obtained pellet was dissolved in 10 mL of a uniform concentration running buffer (20 mM Tris / HCl, pH 7.5) and added to the column.

2.5 Blue HiTrap 5 mL affinity chromatography
A Blue HiTrap 5 mL affinity chromatography column was equilibrated with buffer A (20 mM Tris / HCl, pH 7.5). After loading the pooled active fractions, as determined by molecular sieve chromatography using a hydantoinase assay with Ehrlich reagent (confirmed) and then loading the pooled active fractions, the column was equilibrated with 5 column volumes of buffer B (20 mM). Operate with Tris / HCl, 500 mM NaCl, pH 7.5, then with 2 column volumes of buffer C (20 mM Tris / HCl, pH 7.5, 500 mM NaCl, 1 mM NAD, 1 mM NADP) It was. This affinity chromatography step was used to remove glucose dehydrogenase (not showing activity with respect to glucose and NAD / NADP) by binding to the column.

2.6 Anion exchange chromatography (Mono Q HR 5/5)
The flow-through fraction obtained from the previous affinity chromatography was loaded onto a Mono Q column (diameter 0.5 cm) at a flow rate of 1 mL / min. A linear concentration gradient from running buffer A (20 mM Tris / HCl, pH 7.5) to running buffer B (20 mM Tris / HCl, 750 mM NaCl, pH 7.5) was applied. Fractions were pooled based on the activity of each fraction and the presence of bands in the protein gel.

2.7 Superose 6 Prep Grade 125 mL gel filtration Concentrate pooled active fractions obtained from anion exchange chromatography prior to gel filtration to 1 mL volume (using a Centriprep device with a molecular weight cutoff of 10 kD) and 1 mL The Superose column was loaded at a flow rate of / min. 20 mM Tris / HCl (pH 7.5) was used as a running buffer.

Aliquots from the various purification steps described above were loaded onto a gel and subjected to SDS polyacrylamide electrophoresis (SDS PAGE). The resulting gel was stained with Coomassie Blue, which is shown in FIG. 1, where the lanes correspond to the following from left to right:
1) Molecular weight standards (Precision Plus Protein Marker from BioRad, corresponding to 10, 15, 20, 25, 37, 50, 75, 100, 150 and 250 kD, respectively)
2) Cell-free extract before purification
3) Q-Sepharose FF anion exchange fraction
4) Phenylsepharose FF HIC fraction
5) Superdex Prep Grade 200 gel filtration fraction
6) Blue HiTrap 5 ml affinity chromatography fraction
7) Mono Q HR 5/5 anion exchange fraction
8) Superose 6 prep grade 125 ml gel filtration fraction
9) Molecular weight standard (same as Lane 1)
10) Separate preparation (equivalent to Mono Q HR anion exchange fraction).

  A partially purified protein having a molecular weight of approximately 55 kD was obtained (indicated by the upper arrow in FIG. 1). A second protein of about 35 kD (indicated by the lower arrow in FIG. 1) is glucose dehydrogenase.

  Pool fractions obtained from Superose 6 Prep Grade gel filtration were precipitated with chloroform / methanol and 500 μL formic acid (70%) was added to the precipitate. In order to perform cyanogen bromide cleavage, 1-2 BrCN crystals were added. Bovine serum albumin solution (BSA, 2 mg / mL) was used as a cleavage control. After these two solutions were incubated for 2-3 hours at room temperature, formic acid and cyanogen bromide were removed by a stream of nitrogen. The residue was dissolved in tricine gel sample buffer containing 1% mercaptoethanol and incubated at 95 ° C. for 5 minutes. The resulting sample was loaded onto a tricine gel (10-20% gradient) and blotted onto a PVDF membrane. Peptide fragments on the blot were identified by staining and subjected to N-terminal sequencing.

The following possible peptide sequences were identified:
IELGITGPEGQRLAGPTVL (SEQ ID NO: 1)
IELGITGPEGQRLAGPVL (SEQ ID NO: 2)
IELITGPEGQRLAGPTVL (SEQ ID NO: 3)
IELITGPEGQRLAGPVL (SEQ ID NO: 4)
EEIARARKSGQRVIGEPVAS (SEQ ID NO: 5)
ITGPEGQRLAGP (SEQ ID NO: 7)
The obtained partial sequence is highly homologous to the sequence derived from soybean (Glycine max) of Genbank accession number ACU20291 (SEQ ID NO: 6), but this soybean-derived sequence is attributed to D-hydantoinase. This confirms the hydantoinase properties of the produced protein. SEQ ID NO: 6 is shown below and the homologous region is underlined.

Activity comparison between commercial hydantoinase and isolated hydantoinase
The activities of hydantoinase purchased from Sigma and hydantoinase isolated in Example 2 were compared.

  To measure and compare the specific activity of Sigma enzyme and purified hydantoinase (BP), each of 5 mM hydantoin, 5-phenylhydantoin, or 6-phenyldihydrouracil dissolved in 50 mM Tris HCl pH 7.5, Incubated with various amounts of enzyme at 40 ° C. for 30 minutes.

  The reaction was terminated by adding concentrated hydrochloric acid, and then measured by HPLC.

Table 1 below shows the specific activities of the Sigma enzyme and the enzyme isolated in Example 2. Both enzymes showed the highest activity against 5-phenylhydantoin, followed by hydantoin.

Synthesis of racemic 6-substituted dihydrouracil (overall concept)
The required 6-substituted dihydrouracil 4a-i (Figure 2) was prepared by one of two alternative routes. In Method A (Cabaleiro, MC, Journal of Chemical Research 7: 318-320, 2000), urea is heated at 190 ° C. for 2-4 hours with the appropriate cinnamic acid derivative 7 and then the product is recrystallized, 4a-i was obtained in 45% yield. Method B treated the corresponding racemic β-amino acid with potassium cyanate followed by heating to cause cyclization to give 4a-i in high yield. Method B is known to proceed without racemization and prepares enantiomerically pure (S) -4a and (S) -5a by using enantiomerically pure (S) -6a as starting material. It was also used to

Synthesis of racemic 6-substituted dihydrouracil or precursor molecules (individual synthesis)
a) Synthesis of (±) -6-phenyldihydrouracil of the formula

A mixture of trans-cinnamic acid (10.00 g, 67.5 mmol) and urea (20.25 g, 337.5 mmol) was heated to 210 ° C. over 30 minutes. As a result, a uniform yellow liquid was formed. Urea (2.03 g, 33.8 mmol) was added every hour as the reaction progressed. After stirring for 4 hours, boiling water (80 mL) was added and the reaction mixture was allowed to stir at 95 ° C. for an additional hour. After removing the remaining solid from the reaction mixture by hot filtration, the white precipitate that formed was separated by filtration to give a white solid (6.0 g, 46% yield). R _f 0.3 (EtOAc); δ _H (400 MHz, DMSO-d ₆ ) 2.61 (1H, dd, J = 6.8, 16.1, CH ₂ ), 2.83 (1H, dd, J = 5.8, 16.1, CH ₂ ), 4.64-4.70 (1H, m, CHPh), 7.27-7.41 (5H, m, Ph), 8.01 (1H, bs, CHNHCO), 10.18 (1H, br.s, CONHCO); δ _C (100 MHz, DEPT, DMSO-d ₆ ) 38.6 (CH ₂ ), 50.4 (CHPh), 126.4 (Ph), 128.0 (Ph), 129.0 (Ph), 141.5 (CPh), 154.2 (NHCONH), 170.2 (NHCOCH2); R _t 9.6 and 14.8 min (Chirobiotic T, ammonium acetate buffer pH 5.5: methanol 7: 3, 1 mL / min).

b) Synthesis of trans-methyl cinnamate of the formula

Concentrated sulfuric acid (100 μL) was added to a methanol solution (5 mL) of trans-cinnamic acid (500 mg, 3.4 mmol). After irradiation with microwaves (300 W, 200 ° C.) for 10 minutes, the solvent was removed under reduced pressure. The white solid precipitate was dissolved in ethyl acetate (10 mL) and the organic layer was washed twice with sodium bicarbonate (5 mL) and brine (5 mL), then the solvent was removed in vacuo to give a white crystalline solid (468 mg, 86%). δ _H (400 MHz, DMSO-d ₆ ) 3.73 (3H, s, CH ₃ ), 6.65 (1H, d, J = 16.1, CHCHPh), 7.40-7.46 (3H, m, Ph), 7.67 (2H, d , J = 16.1, CHCHPh), 7.70-7.76 (2H, m, Ph).

c) Synthesis of fluorinated hydroxycinnamoyl of the formula

Dimethylaminosulfur trifluoride (0.31 ml, 2.4 mmol) was added to a solution of trans-cinnamic acid (500 mg, 3.4 mmol) in anhydrous DCM (5 mL) at room temperature under stirring in a nitrogen stream. After stirring for 2 hours, ice-cooled water (5 mL) was added. The organic layer was separated and the aqueous layer was extracted with DCM (5 mL). The DCM layers were combined, washed with ice-cold water (5 mL), dried (MgSO ₄ ) and the solvent removed in vacuo to give a brown oil (451 mg, 88%) that was Used in subsequent reactions without further purification. δ _H (400 MHz, DMSO-d ₆ ): 6.78 (1H, dd, J = 8.0, 16.1 CHC (O) F), 7.45-7.55 (3H, m Ph), 7.81-7.86 (1H, m, Ph) , 7.97 (1H, d, J = 16.0, CHPh) .δC (100 MHz, DEPT, DMSO-d ₆ ) 112.6 (CHCHPc), 129.4 (Ph), 129.6 (Ph), 132.3 (Ph), 133.5 (Ph) , 152.2 (CHPh), 159.1 (C (O) F).

d) Synthesis of (±) -3-fluoro-6-phenyldihydrouracil of the formula

A mixture of p-fluoro-trans-cinnamic acid (1.00 g, 6.67 mmol) and urea (2.00 g, 33.5 mmol) was heated to 210 ° C. over 30 minutes. As a result, a uniform yellow liquid was produced. Additional urea (0.40 g, 6.67 mmol) was added every hour as the reaction proceeded. After stirring for 4 hours, boiling water (20 mL) was added and the reaction mixture was allowed to stir at 95 ° C. for an additional hour. After removing the remaining solid from the reaction mixture by hot filtration, the white precipitate that formed was separated by filtration to give a yellow solid (0.75 g, 60% yield). δ _H (400 MHz, DMSO-d ₆ ) 2.61 (1H, dd, J = 6.8, 16.4, CH ₂ ), 2.84 (1H, dd, J = 5.8, 16.4, CH ₂ ), 4.64-4.70 (1H, m , CHPh), 7.27-7.42 (4H, m, Ph), 8.02 (1H, bs, CHNHCO), 10.19 (1H, br.s, CONHCO); δ _C (100 MHz, DEPT, DMSO-d ₆ ) 38.2 ( CH ₂ ), 50.0 (CHPh), 126.0 (Ph), 127.6 (Ph), 128.5 (Ph), 141.1 (Ph), 153.8 (NHCONH), 169.7 (NHCOCH ₂ ); R _t 10.5 and 15.1 min (Chirobiotic T, (Ammonium acetate buffer, pH 5.5: methanol 7: 3, 1.0 mL / min).

e) Synthesis of (±) -3-bromo-6-phenyldihydrouracil of the formula

A mixture of m-bromo-trans-cinnamic acid (0.80 g, 3.5 mmol) and urea (1.26 g, 21 mmol) was heated to 210 ° C. As a result, a uniform yellow liquid was produced. Additional urea (0.25 g, 4.2 mmol) was added every hour as the reaction proceeded. After stirring for 4 hours, boiling water (20 mL) was added and the reaction mixture was allowed to stir at 95 ° C. for an additional hour. After removing the remaining solid from the reaction mixture by hot filtration, the white precipitate that formed was separated by filtration to give a yellow solid (0.30 g, 32% yield). δ _H (400 MHz, DMSO-d ₆ ) 2.66 (1H, dd, J = 6.8, 17.4, CH ₂ ), 2.84 (1H, dd, J = 5.6, 16.3, CH ₂ ), 4.64-4.74 (1H, m , CHPh), 7.29-7.59 (4H, m, Ph), 8.04 (1H, br.s, CHNHCO), 10.23 (1H, br.s, CONHCO); δ _C (100 MHz, DEPT, DMSO-d ₆ ) 37.8 (CH2), 49.5 (CHPh), 121.1 (Ph), 125.2 (Ph), 129.0 (Ph), 130.5 (Ph), 130.8 (Ph), 143.9 (Ph), 153.7 (NHCONH), 169.6 (NHCOCH2); R _t 16.3 and 25.1 min (Chirobiotic T, ammonium acetate buffer, pH 5.5: methanol 7: 3, 1.0 mL / min).

f) Synthesis of (±) -N-carbamoyl-β-phenylalanine

To a stirred solution of (±) -β-phenylalanine (0.80 g, 4.85 mmol) in hot water (20 mL) was added an aqueous solution (2 mL) of potassium cyanate (0.60 g, 7.40 mmol). After stirring at 90 ° C. for 1 hour, the reaction mixture was allowed to cool to room temperature and then acidified with concentrated hydrochloric acid (pH> 1). On standing, a white precipitate formed which was separated by filtration and recrystallized from boiling water to give a white crystalline solid (0.71 g, 71%). δ _H (400 MHz, DMSO-d ₆ ) 2.63 (2H, CH ₂ CO ₂ H), 5.00 (1H, dd, J = 7.2, 8.7, CHNH), 5.56 (2H, s, NH ₂ ), 6.57 (1H , d, 8.7, CHNH), 7.18-7.34 (5H, m, Ph); δC (100 MHz, DEPT, DMSO-d ₆ ) 41.4 (CH ₂ ), 50.0 (CHPh), 126.3 (Ph), 126.7 (Ph ), 128.1 (Ph), 143.4 (Ph), 157.8 (NHCONH), 172.0 (NHCOCH2); Rt 3.1 and 3.4 min (Chirobiotic T, ammonium acetate buffer, pH 5.5: methanol 7: 3, 1.0 mL / min).

g) Synthesis of (R) -N-carbamoyl-β-phenylalanine of the formula

To a stirred solution of (R) -β-phenylalanine (0.4 g, 2.43 mmol) in hot water (10 mL) was added an aqueous solution (1 mL) of potassium cyanate (0.3 g, 3.70 mmol). After stirring at 90 ° C. for 1 hour, the reaction mixture was allowed to cool to room temperature and then acidified with concentrated hydrochloric acid (pH> 1). On standing, a white precipitate formed which was separated by filtration and recrystallized from boiling water to give a white crystalline solid (0.30 g, 60%). δ _H and δ _C analyzes are identical to (±) -46; R _t 3.4 min (Chirobiotic T, ammonium acetate buffer, pH 5.5: methanol 7: 3, 1.0 mL / min).

h) Synthesis of (±) -6-phenyldihydrouracil (of the formula below) from (±) -N-carbamoyl-β-phenylalanine

  A solution of (±) -N-carbamoyl-β-phenylalanine (100 mg, 0.48 mmol) in concentrated hydrochloric acid (5 mL) was stirred at 135 ° C. for 4 hours. After that time, the volume of the reaction mixture had decreased by 75%, but the reaction mixture was cooled to room temperature. Upon standing a white precipitate formed which was separated by filtration to give a white crystalline solid (50 mg, 55%): analysis identical to (±) -45.

i) Synthesis of (S) -6-phenyldihydrouracil of the formula

To a stirred solution of (S) -β-phenylalanine (100 mg, 0.60 mmol) in hot water (2 mL) was added an aqueous solution (0.5 mL) of potassium cyanate (75 mg, 0.93 mmol). After stirring at 90 ° C. for 1 hour, concentrated hydrochloric acid (2 mL) was added, and the reaction solution was stirred at 135 ° C. for 2 hours. After that time, the volume of the mixture had decreased by 75%, but the reaction mixture was cooled to room temperature. Upon standing, a white precipitate formed, which was separated by filtration to give a white crystalline solid (55 mg, 48%); analysis of δ _H and δ _C is identical to (±) -45; R _t 9.6 min (Chirobiotic T, ammonium acetate buffer pH 5.5: methanol 7: 3, 1 mL / min).

Separation of precipitates and hydantoinase reaction products
The binding of 6-PDHU and NCBPA to Q Sepharose and DEAE Sepharose was tested in a microcap (Eppendorf). After washing 1 mL Q Sepharose or DEAE Sepharose (commercial ethanol suspension) with water (3 x 1 mL), add a solution (1 mL) containing 2.5 mM NCBPA and 2.5 mM 6-PDHU, An aliquot of the supernatant was removed after vortexing the cap. The percentage of NCBPA and 6PDHU in the supernatant was determined by rpHPLC and is shown in Table 2.

  The binding to the two resins is rapid, with Q Sepharose binding to N-carbamoyl-β-phenylalanine less than DEAE and more binding to 6-phenyldihydrouracil than DEAE, so DEAE is more preferred for separation. It becomes an anion exchange resin.

Chiral reversed phase HPLC
The following columns, buffers and running conditions were used for chiral rpHPLC:
^{Astec Chirobiotic TM T 25 cm x 4.6} mm, 5μm (Sigma-Aldrich, St. Louis, USA)
Ammonium acetate buffer (pH 5.5): methanol, 70:30, 25 ° C., 0.5 mL / min.

Enzyme, substrate and product separation columns by DEAE chromatography were prepared from inverted syringes, filled with glass wool and packed with 10 column volumes of DEAE (4 mL) washed with dH ₂ O. After equilibration with 200 mM ammonium acetate buffer (pH 4), a synthetic mixture of NCBPA (2.5 mM), 6PDHU (2.5 mM) and enzyme (7 mg / mL) in H ₂ O was applied to the column, and the flow-through fraction was rpHPLC ( 6PDHU / NCBPA detection) and Bradford assay (enzyme detection). After all 6PDHU was washed from the column, NCBPA was eluted from the column using 200 mM ammonium acetate buffer (pH 8.17). The results are shown in FIG. 3 (squares represent 6-PDHU; diamonds represent NCBPA, triangles represent enzymes; 6-PDHU and NCBPA are in units of mM and enzymes are in units of mg / L). At pH 4, a clear separation of NCBPA and 6PDHU was observed, enabling a 90% yield of NCBPA.

Hydrolysis of racemic 6-substituted dihydrouracil catalyzed by hydantoinase Introduction of (±) -6-PDHU (racemic 6-phenyldihydrouracil) catalyzed by hydantoinase (purchased from Sigma, St. Louis, USA) Hydrolysis (4a in Figure 2: R = Ph) was studied as a model system. The reaction was carried out at a substrate concentration of 5 mM in Tris buffer [pH 7.5] and was monitored by reverse phase chiral HPLC as described in Example 7, which determined the degree of conversion in the reaction and the unreacted dihydrouracil 4a and N- Both of the enantiomeric excess of carbamoyl derivative 5 could be measured simultaneously (see also FIG. 4, scheme 2).

We focused on the following observations:
(i) The hydantoinase enzyme was found to be highly selective for the (S) -enantiomer of 6-PDHU (4a) with an E value> 100. The absolute configuration of the product (S) -N-carbamoyl-β-phenylalanine [(S) -NCBPA] and unreacted substrate (R) -6-DPHU was determined relative to a standard;
(ii) Despite the high (S) enantioselectivity observed, the conversion did not proceed to 50% even after extending the reaction time. All reactions gave an equilibrium concentration of 6-PDHU: (S) -NCBPA = 51: 49. The enantiomeric excess of (S) -NCBPA exceeded 96% during the entire observation period. FIG. 5 shows the results obtained from a 25 mL reaction in a small reactor, where the diamonds represent NCBPA concentration [mM], the squares represent 6-DPHU concentration [mM], and the x symbol represents enantiomeric excess. Expresses the rate [%].

(iii) The reverse reaction, i.e. cyclization of N-carbamoyl-β-phenylalanine, was found to have a significant ratio to hydrolysis, which was also highly (S) -selective and (R) -Little cyclization of enantiomers was observed. FIG. 6 shows the conversion of (+/-)-, (S)-and (R) -NCBPA with and without the enzyme, with the black or white diamonds connected by solid lines + // -NCBPA (without enzyme) or +/- NCBPA (with enzyme), black or white squares connected by solid lines are (S) -NCBPA (without enzyme) or (S) -NCBPA (enzyme, respectively) The black triangle or white triangle connected with a solid line represents (R) -NCBPA (without enzyme) or (R) -NCBPA (with enzyme), respectively.

PH dependence of chiral hydrolysis of 6-PDHU
6-PDHU was incubated at 50 ° C. for 24 hours in Tris buffer at various pH values. Considerable background hydrolysis of 6-PDHU occurred strongly depending on pH and buffer. 0.5% background hydrolysis was observed in pH 7.0 Tris buffer. At pH 9, background hydrolysis reached approximately 20% (Figure 7).

PH dependence of hydantoinase activity.
6-PDHU was incubated at various pH values (up to 8 hours) in phosphate buffer (0.1 M, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, adjusted to the appropriate pH) or Tris buffer. Sigma D-hydantoinase 7 g / L). The highest conversion was observed in the range from about pH 7.5 to about pH 9.0 (FIG. 8, squares represent phosphate buffer and diamonds represent Tris buffer).

In additional experiments, conversion was measured over time at various pH for phosphate or Tris buffer. 5 mM 6-PDHU was incubated for 22 hours in phosphate buffer (pH 6.0, 7.0, 7.5, 8.0) and Tris buffer (pH 7.5). The results are shown in FIG. 9 (diamonds, squares, triangles and crosses represent phosphate buffer at pH 6, pH 7, pH 7.5 or pH 8, respectively, and black circles represent Tris buffer at pH 7.5) and Table 3 (ee = mirror image) Body excess rate [%]).

  The conversion proceeds with a high enantiomeric excess of the desired substrate at pH 6.0 in phosphate buffer, but the conversion is considerably lower compared to conversion at pH values from 7.00 to 8.00.

Substrate specificity of azuki bean (Vigna angularis) hydantoinase As described in Example 4, various substrates were synthesized and subjected to a conversion reaction catalyzed by hydantoinase (Tris buffer (pH 7.5, 0.1M, 1.0 mL), D -Hydantoinase (Sigma, 7g / L), substrate (5mM solution, aromatic substituents (4a, 4b, 4c) unless dissolved in buffer beforehand, substrate is dissolved in 100μL DMSO in the case of aromatic substituents) Added to 900 μL buffer)]. The reaction is shown in FIG. 10 and the results are shown in Table 4, whose E value is defined as follows:
E value = (ln (1- [product] * (1 + ee))) / (ln (1- [product] * (1-ee)))

Effects of organic cosolvents on the conversion catalyzed by hydantoinase
Biotransformation of 5 mM 6-PDHU is performed on a 5 mL scale in 2% DMSO containing buffer and buffer saturated with MTBE (approximately 5% by volume) or butan-2-ol (approximately 15% by volume) did. The results are shown in FIG. 11, where diamonds represent buffers, squares represent 2% DMSO-containing buffers, triangles represent MTBE-containing buffers, and crosses represent butan-2-ol-containing buffers. Neither DMSO nor MTBE had a significant effect on the conversion, but butan-2-ol reduced the amount of NCBPA at the concentrations used.

Effect of enzyme content on conversion and enantiomeric excess
Three reaction settings were performed in parallel at a temperature of 50 ° C. with a total reaction volume of 5 mL Tris buffer, pH 7.5 each containing 5 mM 6-PDHU. In one reaction, the normal enzyme amount (7 g / L) was used, while the other settings contained 0.25 times equivalent (1.75 g / L) and 4 times equivalent (28 g / L), respectively. The production of N-carbamoyl-β-phenylalanine (NCBPA) and the enantiomeric excess of the S-enantiomer were measured at various time points. The results are shown in FIG. 12, where the column, shaded with vertical, diagonal, or horizontal lines, shows the concentration of NCBPA obtained in the presence of 0.25-fold, 1-fold, or 4-fold equivalent enzyme, respectively. The diamonds, squares and triangles represent the corresponding enantiomeric excess in the presence of 0.25, 1 or 4 times the equivalent amount of enzyme, respectively.

Scale-up of reactions catalyzed by hydantoinase
In three independent biotransformations, 20 mM 6-PDHU was incubated at 50 ° C. in the presence of 175 mg hydantoinase with a total reaction volume of 25 mM buffer (Tris buffer pH 7.5). The pH of the buffer was adjusted after the addition of 6-PDHU and kept constant during the reaction process by automatic titration of NaOH. To measure the concentration of NCBPA and the enantiomeric excess of the S-enantiomer, aliquots were divided at various time points. The results are shown in FIGS. 13 and 14, respectively. In FIG. 13, diamonds, squares, and triangles represent reactor run 1, reactor run 2, and reactor run 3, respectively. In FIG. 14, diamonds, squares and triangles represent the enantiomeric excess observed in Reactor Run 1, Reactor Run 2, and Reactor Run 3, respectively, and the solid, dashed, and dotted lines represent Reactor Run 1, Reactor Run 2, And represents the most applicable curve of enantiomeric excess observed in Reactor Run 3.

Summary of SEQ ID NO: SEQ ID NO: 1-Peptide sequence obtained from isolated hydantoinase SEQ ID NO: 2-Peptide sequence obtained from isolated hydantoinase SEQ ID NO: 3-Peptide sequence sequence obtained from isolated hydantoinase SEQ ID NO: 5-Peptide sequence obtained from isolated hydantoinase SEQ ID NO: 5-Peptide sequence obtained from isolated hydantoinase SEQ ID NO: 6-Hydantoinase from soybean (Glycine max) as a sequence reference SEQ ID NO: 7-Isolation Partial peptide sequence abbreviations obtained from purified hydantoinases
NCBPA-N-carbamoyl-β-phenylalanine
6PDHU-6-Phenyldihydrouracil

Claims (17)

A method for producing a β-amino acid precursor with a biocatalyst comprising at least one general formula (I),

[Wherein R ¹ and R ² are independently of each other,
hydrogen,
An optionally substituted linear or branched lower alkyl group,
A linear or branched lower alkenyl group, which may be substituted,
An optionally substituted cyclic alkyl group,
An optionally substituted monocyclic or polycyclic aryl group,
A monocyclic or polycyclic heteroaryl group, which may be substituted,
A linear or branched alkoxy group which may be substituted,
An amino group,
A linear or branched alkylamino group which may be substituted,
An optionally substituted linear or branched alkylthio group,
An optionally substituted linear or branched acyl group,
Selected from carboxyl group or aldehyde group]
A substrate represented by
Wherein the substrate is present in stereoisomerically pure form or as a mixture of stereoisomers, the stereoisomerically pure form or mixture of stereoisomers may be a salt,
In the presence of at least one enzyme that catalyzes the hydrolysis of the hydantoin and / or dihydropyrimidine ring,
Thereby, a β-amino acid precursor of the general formula (II),

[Wherein R ¹ and R ² are the same as above]
Generating,
A method comprising
At least one enzyme used in the method is
(a) has a hydantoinase and / or dihydropyrimidinase activity, obtained from azuki bean (Vigna angularis), viewed at least Tsuo含amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7, about as determined by SDS-PAGE An enzyme exhibiting a molecular weight of 55 kD, wherein the term “about” is an enzyme exhibiting a ± 5% variation of the indicated value ;
Or
(b) has 1-10 amino acid additions, substitutions, deletions and / or inversions to the enzyme of (a) and has at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7 Said method characterized in that it is an enzyme comprising an amino acid sequence and having hydantoinase and / or dihydropyrimidinase activity. The β-amino acid precursor of formula (II), the corresponding β-amino acid of formula (III)

[Wherein R ¹ and R ² have the same meaning as described in claim 1]
The method of claim 1, further comprising converting to:   3. The method of claim 2, wherein the conversion of the beta amino acid precursor occurs at acidic pH or in the presence of a carbamoylase. The method according to claim ¹ , wherein R ² is H and R ¹ is an optionally substituted aryl group.   The method according to claim 1, wherein the reaction is carried out in a Tris buffer or borate buffer reaction mixture.   6. The reaction according to any one of claims 1-5, wherein the reaction is carried out at a pH of about 7.0 to about 11.0, wherein the term "about" indicates a ± 5% variation in the indicated value. Method.   The reaction is carried out in the presence of about 1% to about 20% dimethyl sulfoxide, wherein the term "about" indicates a ± 5% variation of the indicated value. The method according to any one of the above.   The reaction is carried out at a temperature of about 30 ° C. to about 60 ° C., wherein the term “about” indicates a variation of ± 5% of the indicated value according to any one of claims 1-7. The method described.   9. The reaction according to any one of claims 1 to 8, wherein the reaction is carried out from about 1 hour to about 25 hours, wherein the term "about" indicates a ± 5% variation in the indicated value. Method.   10. A method according to any one of claims 1 to 9, wherein the at least one substrate is dihydrouracil monosubstituted at the 5 or 6 position.   11. The method according to any one of claims 1 to 10, wherein the at least one enzyme comprises the amino acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 7.   12. A method according to any one of the preceding claims, wherein the reaction is carried out in a Tris buffer reaction mixture.   13. The reaction according to any one of claims 1-12, wherein the reaction is carried out at a pH of about 7.5 to about 8.0, wherein the term "about" indicates a ± 5% variation in the indicated value. Method.   14. The reaction is performed in the presence of about 10% dimethyl sulfoxide, wherein the term “about” indicates a ± 5% variation in the indicated value. the method of.   The reaction is carried out at a temperature of about 40 ° C. to about 50 ° C., wherein the term “about” indicates a variation of ± 5% of the indicated value according to any one of claims 1-14. The method described.   The reaction is performed from about 4 hours to about 5 hours, wherein the term "about" indicates a ± 5% variation in the indicated value. Method.   At least one substrate is selected from the group consisting of 6-phenyldihydrouracil, 4-fluoro-6-phenyldihydrouracil, 3-bromo-6-phenyldihydrouracil, 5-methyldihydrouracil, and 6-methyldihydrouracil. The method according to any one of claims 1 to 16.

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